DNA'Protein Interactions: Principles and Protocols

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Methods in Molecular Biology

TM

VOLUME 148

DNA–Protein Interactions Principles and Protocols SECOND EDITION

Edited by

Tom Moss PO

LII

TF

IIH

HUMANA PRESS

DNA–Protein Interactions

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Preface DNA–protein interactions are fundamental to the existence of life forms, providing the key to the genetic plan as well as mechanisms for its maintenance and evolution. The study of these interactions is therefore fundamental to our understanding of growth, development, differentiation, evolution, and disease. The manipulation of DNA–protein interactions is also becoming increasingly important to the biotechnology industry, permitting among other things the reprogramming of gene expression. The success of the first edition of DNA– Protein Interactions; Principles and Protocols was the result of Dr. G. Geoff Kneale's efforts in bringing together a broad range of relevant techniques. In producing the second edition of this book, I have tried to further increase this diversity while presenting the reader with alternative approaches to obtaining the same information. A major barrier to the study of interactions between biological macromolecules has always been detection and hence the need to obtain sufficient material. The development of molecular cloning and subsequently of protein overexpression systems has essentially breached this barrier. However, in the case of DNA–protein interactions, the problem of quantity and hence of detection is often offset by the high degree of selectivity and stability of DNA– protein interactions. DNA–protein binding reactions will often go to near completion at very low component concentrations even within crude protein extracts. Thus, although many techniques described in this volume were initially developed to study interactions between highly purified components, these same techniques are often just as applicable to the identification of novel DNA–protein interactions within systems as undefined as a whole cell extract. In general, these techniques use a DNA rather than a protein detection system because the former is more sensitive. Radiolabeled DNA fragments are easily produced by a range of techniques commonly available to molecular biologists. DNA–protein complexes may be studied at three distinct levels—at the level of the DNA, of the protein, and of the complex. At the level of the DNA, the DNA binding site may be delimited and exact base sequence requirements defined. The DNA conformation can be studied and the exact bases contacted

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Methods in Molecular BIOLOGY

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John M. Walker, Series Editor 178.`Antibody Phage Display: Methods and Protocols, edited by Philippa M. O’Brien and Robert Aitken, 2001 177. Two-Hybrid Systems: Methods and Protocols, edited by Paul N. MacDonald, 2001 176. Steroid Receptor Methods: Protocols and Assays, edited by Benjamin A. Lieberman, 2001 175. Genomics Protocols, edited by Michael P. Starkey and Ramnath Elaswarapu, 2001 174. Epstein-Barr Virus Protocols, edited by Joanna B. Wilson and Gerhard H. W. May, 2001 173. Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J. Vogel, 2001 172. Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J. Vogel, 2001 171. Proteoglycan Protocols, edited by Renato V. Iozzo, 2001 170. DNA Arrays: Methods and Protocols, edited by Jang B. Rampal, 2001 169. Neurotrophin Protocols, edited by Robert A. Rush, 2001 168. Protein Structure, Stability, and Folding, edited by Kenneth P. Murphy, 2001 167. DNA Sequencing Protocols, Second Edition, edited by Colin A. Graham and Alison J. M. Hill, 2001 166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001 165. SV40 Protocols, edited by Leda Raptis, 2001 164. Kinesin Protocols, edited by Isabelle Vernos, 2001 163. Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R. Mitchelson and Jing Cheng, 2001 162. Capillary Electrophoresis of Nucleic Acids, Volume 1: Introduction to the Capillary Electrophoresis of Nucleic Acids, edited by Keith R. Mitchelson and Jing Cheng, 2001 161. Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001 160. Nuclease Methods and Protocols, edited by Catherine H. Schein, 2001 159. Amino Acid Analysis Protocols, edited by Catherine Cooper, Nicole Packer, and Keith Williams, 2001 158. Gene Knockoout Protocols, edited by Martin J. Tymms and Ismail Kola, 2001 157. Mycotoxin Protocols, edited by Mary W. Trucksess and Albert E. Pohland, 2001 156. Antigen Processing and Presentation Protocols, edited by Joyce C. Solheim, 2001 155. Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000 154. Connexin Methods and Protocols, edited by Roberto Bruzzone and Christian Giaume, 2001 153. Neuropeptide Y Protocols , edited by Ambikaipakan Balasubramaniam, 2000 152. DNA Repair Protocols: Prokaryotic Systems, edited by Patrick Vaughan, 2000 151. Matrix Metalloproteinase Protocols, edited by Ian M. Clark, 2001 150. Complement Methods and Protocols, edited by B. Paul Morgan, 2000 149. The ELISA Guidebook, edited by John R. Crowther, 2000 148. DNA–Protein Interactions: Principles and Protocols (2nd ed.), edited by Tom Moss, 2001

147. Affinity Chromatography: Methods and Protocols, edited by Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, and Wolfgang Berthold, 2000 146. Mass Spectrometry of Proteins and Peptides, edited by John R. Chapman, 2000 145. Bacterial Toxins: Methods and Protocols, edited by Otto Holst, 2000 144. Calpain Methods and Protocols, edited by John S. Elce, 2000 143. Protein Structure Prediction: Methods and Protocols, edited by David Webster, 2000 142. Transforming Growth Factor-Beta Protocols, edited by Philip H. Howe, 2000 141. Plant Hormone Protocols, edited by Gregory A. Tucker and Jeremy A. Roberts, 2000 140. Chaperonin Protocols, edited by Christine Schneider, 2000 139. Extracellular Matrix Protocols, edited by Charles Streuli and Michael Grant, 2000 138. Chemokine Protocols, edited by Amanda E. I. Proudfoot, Timothy N. C. Wells, and Christine Power, 2000 137. Developmental Biology Protocols, Volume III, edited by Rocky S. Tuan and Cecilia W. Lo, 2000 136. Developmental Biology Protocols, Volume II, edited by Rocky S. Tuan and Cecilia W. Lo, 2000 135. Developmental Biology Protocols, Volume I, edited by Rocky S. Tuan and Cecilia W. Lo, 2000 134. T Cell Protocols: Development and Activation, edited by Kelly P. Kearse, 2000 133. Gene Targeting Protocols, edited by Eric B. Kmiec, 2000 132. Bioinformatics Methods and Protocols, edited by Stephen Misener and Stephen A. Krawetz, 2000 131. Flavoprotein Protocols, edited by S. K. Chapman and G. A. Reid, 1999 130. Transcription Factor Protocols, edited by Martin J. Tymms, 2000 129. Integrin Protocols, edited by Anthony Howlett, 1999 128. NMDA Protocols, edited by Min Li, 1999 127. Molecular Methods in Developmental Biology: Xenopus and Zebrafish, edited by Matthew Guille, 1999 126. Adrenergic Receptor Protocols, edited by Curtis A. Machida, 2000 125. Glycoprotein Methods and Protocols: The Mucins, edited by Anthony P. Corfield, 2000 124. Protein Kinase Protocols, edited by Alastair D. Reith, 2001 123. In Situ Hybridization Protocols (2nd ed.), edited by Ian A. Darby, 2000 122. Confocal Microscopy Methods and Protocols, edited by Stephen W. Paddock, 1999 121. Natural Killer Cell Protocols: Cellular and Molecular Methods, edited by Kerry S. Campbell and Marco Colonna, 2000 120. Eicosanoid Protocols, edited by Elias A. Lianos, 1999 119. Chromatin Protocols, edited by Peter B. Becker, 1999 118. RNA–Protein Interaction Protocols, edited by Susan R. Haynes, 1999 117. Electron Microscopy Methods and Protocols, edited by M. A. Nasser Hajibagheri, 1999 116. Protein Lipidation Protocols, edited by Michael H. Gelb, 1999 115. Immunocytochemical Methods and Protocols (2nd ed.), edited by Lorette C. Javois, 1999

Methods in Molecular BIOLOGY

DNA–Protein Interactions Principles and Protocols Second Edition

Edited by

Tom Moss Centre de Recherche en Cancérologie de l’Université Laval, Centre Hopital Universitaire de Québec et Départment de biologie médicale, Université Laval, Québec, QC, Canada

Humana Press

Totowa, New Jersey

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©2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology™ is a trademark of The Humana Press Inc. Cover design by Patricia F. Cleary Cover Figure: A structural model for the RNA polymerase II open complex as determined by site-specific protein-DNA UV photo-cross-linking. Promoter DNA is wrappedaround RNA polymerase II (POL II), allowing contacts by the Xeroderma Pigmentosum Group B (XPB) helicase of transcription factor TFIIH to the template strand of the melted DNA duplex immediately upstream of the transcription initiation site. Transcription factors TBP, TFIIB, TFIIE and TFIIF, which are part of the complex, are not shown. For additional details, see Douziech et al. (2000) Mol. Cell. Biol. 20: 8168-8177. Cover image kindly provided by Dr. Benoit Coulombe, Univerity of Sherbrooke, Quebec, Canada; Imaging: MOLECULAR IMAGE, University ofSherbrooke, Quebec, Canada. Production Editor: Jason Runnion The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected] or visit our Website at www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-625-1/01 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data DNA-protein interactions : principles and protocols / edited by Tom Moss.--2nd ed. p. cm.--(Methods in molecular biology ; v. 148) Includes bibliographical references and index. ISBN 0-89603-625-1 (hc : alk. paper) -- ISBN 0-89603-671-5 (pbk.: alk. paper) 1. DNA-protein interactions. I. Moss, Tom. II. Series. QP624.75.P74 D57 2001 572.8'6--dc21

00-054100 CIP

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Preface

by the protein identified. At the protein level, the protein species binding a given DNA sequence can be identified. The amino acids contacting DNA and the protein surface facing the DNA may be defined and the amino acids essential to the recognition process can be identified. Furthermore, the protein’s tertiary structure and its conformational changes on complex formation can be studied. Finally, global parameters of a DNA–protein complex such as stoichiometry, the kinetics of its formation and dissociation, its stability, and the energy of interaction can be measured. Filter binding, electrophoretic mobility shift assay (EMSA/gel shift), DNaseI footprinting, and Southwestern blotting have been the most commonly used techniques to identify potentially interesting DNA target sites and to define the proteins that bind them. For example, gel shift or footprinting of a cloned gene regulation sequence by proteins in a crude cell extract may define binding activities for a given DNA sequence that correlates with gene expression or silencing. These techniques can be used as an assay during subsequent isolation of the protein(s) responsible. Interference assays, SELEX, and more refined footprinting techniques, such as hydroxy radical footprinting and DNA bending assays, can then be used to study the DNA component of the DNA–protein complex, whereas the protein binding surface can be probed by amino acid side chain modification, DNA–protein crosslinking, and of course by the production of protein mutants. Genetic approaches have also opened the way to engineer proteins recognizing chosen DNA targets. DNA–protein crosslinking has in recent years become a very important approach to investigate the relative positions of proteins in multicomponent protein–DNA complexes such as the transcription initiation complex. Here, crosslinkable groups are incorporated at specific DNA sequences and these are used to map out the “positions” of different protein components along the DNA. Extension of this technique can also allow the mapping of the crosslink within the protein sequence. Similar data can be obtained by incorporating crosslinking groups at known sites within the protein and then identifying the nucleotides targeted. Once the basic parameters of a DNA–protein interaction have been defined, it is inevitable that a deeper understanding of the driving forces behind the DNA–protein interaction and the biological consequences of its formation will require physical and physicochemical approaches. These can be either static or dynamic measurements, but most techniques have been developed to deal with steady-state situations. Equilibrium constants can be obtained by surface plasmon resonance, by spectroscopic assays that differentiate complexed and uncomplexed components, and, for more stable products, by footprinting and gel shift. Spectroscopy can also give specific answers about

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the conformation of proteins and any conformational changes they undergo on interacting with DNA as well as providing a rapid quantitative measure of complex formation. Microcalorimetry gives a global estimation of the forces stabilizing a given complex. Static pictures of protein–DNA interactions can be obtained by several techniques. At atomic resolution, X-ray crystallography, and nuclear magnetic resonance (NMR) studies require large amounts of highly homogeneous material. Lower resolution images can be obtained by electron and, more recently, by atomic force microscopies. Large multiprotein complexes are generally beyond the scope of NMR or even of X-ray crystallography. These are therefore more often studied using the electron microscope, either in a direct imaging mode or via the analysis of data obtained from 2D pseudocrystalline arrays. Dynamic measurements of complex formation or dissociation can be obtained by biochemical techniques when the DNA–protein complexes have half-lives of several minutes to several hours. For footprinting and crosslinking, a general rule is that the complexes should be stable for a time well in excess of the proposed period of the enzymatic or chemical reaction. For gel shift, the complex half-life should at least approach that of the time of gel migration, although the cage effect may tend to stabilize the complex within the gel matrix, extending the applicability of this technique. More rapid assembly kinetics, multistep assembly processes, and short-lived DNA–protein complexes require much more rapid techniques such as UV laser-induced crosslinking, surface plasmon resonance, and spectroscopic assays. UV-laser induced DNA– protein crosslinking is a promising development because it potentially permits the kinetics of complex assembly to be followed both in vitro and in vivo. When I decided to edit a second edition of the present volume, I was of course aware of the limitations of many of the more commonly used techniques. But as I read the various chapters I realized that each technique was at least as much limited by the conditions necessary for the probing reaction itself as by the type of information the probe could deliver. This is perhaps most evident for in vivo applications, which require agents that can easily enter cells, e.g., DMS and potassium permanganate are able to penetrate cells while DNaseI and DEPC are either too large or insufficiently water soluble to enter cells unaided. (Appendix II presents a summary of the activities and applications of the various DNA modification and cleavage reagents described in this book.) Gel shift assays are limited by the finite range of useable electrophoresis conditions. Because buffers must have low conductance, the KCl or NaCl solutions typically used for DNA–protein binding reactions are generally inappropriate. (Appendix I contains a list of the different gel shift conditions described in various chapters of this book.) Thus, it is often as

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important to choose a technique appropriate to the conditions under which one wishes to observe the DNA–protein interaction as it is to choose the appropriate probing activity. The present volume attempts to bring together a broad range of techniques used to study DNA–protein interactions. Such a volume can never be complete nor definitive, but I hope this book will provide a useful source of technical advice for molecular biologists. Its preparation required the cooperation of many people. In particular I would like to thank all the authors for their very significant efforts. Thanks are also due to John Walker for his encouragement and to the previous editor Geoff Kneale and to Craig Adams of Humana Press for their help. I also thank Margrit and Peter Wittwer for providing space in the Pfarrhaus of the Predigerkirche, Zürich, where much of the chapter editing was done, and Bernadette for her patience, understanding, corrections, and advice.

Tom Moss

Contents Preface ............................................................................................................. v Contributors ................................................................................................... xiii 1 Filter-Binding Assays Peter G. Stockley ................................................................................... 1 2 Electrophoretic Mobility Shift Assays for the Analysis of DNA–Protein Interactions Marc-André Laniel, Alain Béliveau, and Sylvain L. Guérin ............ 13 3 DNase I Footprinting Benoît Leblanc and Tom Moss .......................................................... 31 4 Footprinting with Exonuclease III Willi Metzger and Hermann Heumann ............................................... 39 5 Hydroxyl Radical Footprinting Evgeny Zaychikov, Peter Schickor, Ludmilla Denissova, and Hermann Heumann .................................................................. 49 6 The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA Brenda F. Kahl and Marvin R. Paule ................................................. 63 7 Footprinting DNA–Protein Interactions in Native Polyacrylamide Gels by Chemical Nucleolytic Activity of 1,10-Phenanthroline-Copper Athanasios G. Papavassiliou ............................................................. 77 8 Uranyl Photofootprinting Peter E. Nielsen .................................................................................. 111 9 Osmium Tetroxide Modification and the Study of DNA–Protein Interactions James A. McClellan ........................................................................... 121 10 Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots Athanasios G. Papavassiliou ........................................................... 135 11 Diffusible Singlet Oxygen as a Probe of DNA Deformation Malcolm Buckle and Andrew A. Travers ........................................ 151

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12 Ultraviolet-Laser Footprinting Johannes Geiselmann and Frederic Boccard ............................... 161 13 In Vivo DNA Analysis Régen Drouin, Jean-Philippe Therrien, Martin Angers, and Stéphane Ouellet.................................................................... 175 14 Identification of Protein–DNA Contacts with Dimethyl Sulfate: Methylation Protection and Methylation Interference Peter E. Shaw and A. Francis Stewart ............................................ 221 15 Ethylation Interference Iain W. Manfield and Peter G. Stockley........................................... 229 16 Hydroxyl Radical Interference Peter Schickor, Evgeny Zaychikov, and Hermann Heumann ...... 245 17 Identification of Sequence-Specific DNA-Binding Proteins by Southwestern Blotting Simon Labbé, Gale Stewart, Olivier LaRochelle, Guy G. Poirier, and Carl Séguin .................................................. 255 18 A Competition Assay for DNA Binding Using the Fluorescent Probe ANS Ian A. Taylor and G. Geoff Kneale ................................................... 265 19 Site-Directed Cleavage of DNA by Linker Histone Protein-Fe(II) EDTA Conjugates David R. Chafin and Jeffrey J. Hayes ............................................. 275 20 Nitration of Tyrosine Residues in Protein–Nucleic Acid Complexes Simon E. Plyte .................................................................................... 291 21 Chemical Modification of Lysine by Reductive Methylation: A Probe of Residues Involved in DNA Binding Ian A. Taylor and Michelle Webb ..................................................... 301 22 Limited Proteolysis of Protein–Nucleic Acid Complexes Simon E. Plyte and G. Geoff Kneale................................................ 315 23 Ultraviolet Crosslinking of DNA–Protein Complexes via 8-Azidoadenine Rainer Meffert, Klaus Dose, Gabriele Rathgeber, and Hans-Jochen Schäfer ............................................................ 323 24 Site-Specific Protein–DNA Photocrosslinking: Analysis of Bacterial Transcription Initiation Complexes Nikolai Naryshkin, Younggyu Kim, Qianping Dong, and Richard H. Ebright ................................................................. 337

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25 Site-Directed DNA Photoaffinity Labeling of RNA Polymerase III Transcription Complexes Jim Persinger and Blaine Bartholomew ......................................... 363 26 Use of Site-Specific Protein–DNA Photocrosslinking to Analyze the Molecular Organization of the RNA Polymerase II Initiation Complex François Robert and Benoît Coulombe .......................................... 383 27 UV Laser-Induced Protein–DNA Crosslinking Stefan I. Dimitrov and Tom Moss .................................................... 395 28 Plasmid Vectors for the Analysis of Protein-Induced DNA Bending Christian Zwieb and Sankar Adhya ................................................. 403 29 Engineering Nucleic Acid-Binding Proteins by Phage Display Mark Isalan and Yen Choo ................................................................ 417 30 Genetic Analysis of DNA–Protein Interactions Using a Reporter Gene Assay in Yeast David R. Setzer, Deborah B. Schulman, and Michael J. Bumbulis .............................................................. 431 31 Assays for Transcription Factor Activity Virgil Rhodius, Nigel Savery, Annie Kolb, and Stephen Busby ....................................................................... 451 32 Assay of Restriction Endonucleases Using Oligonucleotides Bernard A. Connolly, Hsiao-Hui Liu, Damian Parry, Lisa E. Engler, Michael R. Kurpiewski, and Linda Jen-Jacobson ............................................................. 465 33 Analysis of DNA–Protein Interactions by Intrinsic Fluorescence Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale ....... 491 34 Circular Dichroism for the Analysis of Protein–DNA Interactions Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale ....... 503 35 Calorimetry of Protein–DNA Complexes and Their Components Christopher M. Read and Ilian Jelesarov ....................................... 511 36 Surface Plasmon Resonance Applied to DNA–Protein Complexes Malcolm Buckle .................................................................................. 535 37 Reconstitution of Protein–DNA Complexes for Crystallization Rachel M. Conlin and Raymond S. Brown ..................................... 547 38 Two-Dimensional Crystallization of Soluble Protein Complexes Patrick Schultz, Nicolas Bischler, and Luc Lebeau ...................... 557

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39 Atomic Force Microscopy of DNA and Protein–DNA Complexes Using Functionalized Mica Substrates Yuri L. Lyubchenko, Alexander A. Gall, and Luda S. Shlyakhtenko ........................................................... 569 40 Electron Microscopy of Protein–Nucleic Acid Complexes: Uniform Spreading of Flexible Complexes, Staining with a Uniform Thin Layer of Uranyl Acetate, and Determining Helix Handedness Carla W. Gray ..................................................................................... 579 41 Scanning Transmission Electon Microscopy of DNA–Protein Complexes Joseph S. Wall and Martha N. Simon .............................................. 589 42 Determination of Nuleic Acid Recognition Sequences by SELEX Philippe Bouvet .................................................................................. 603 43 High DNA–Protein Crosslinking Yield with Two-Wavelength Femtosecond Laser Irradiation Christoph Russmann, Rene Beigang, and Miguel Beato ............. 611 Appendices: Appendix I: EMSA/Gel Shift Conditions .............................................. 617 Appendix II: DNA-Modification/Cleavage Reagents ........................... 619 Index ............................................................................................................ 621

Contributors SANKAR ADHYA • Laboratory of Molecular Biology, National Institutes of Health, NCI, Bethesda, MD MARTIN ANGERS • Division de Pathologie, Department de Biologie Médicale, Université Laval, et Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche, Pavilion Saint-Francois d’Assise, Québec, Canada BLAINE BARTHOLOMEW • Department of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University, Carbondale, IL MIGUEL BEATO • Insitute für Molekularbiologie und Tumorforshung, Philipps-Universität Marburg, Marburg, Germany RENE BEIGANG • Fachbereich Physik, Universität Kaiserlautern, Germany ALAIN BÉLIVEAU • Laboratory of Molecular Endocrinologie, Centre Hopitalier Universitaire de Québec, Université Laval, Québec, Canada NICOLAS BISCHLER • Faculté de Médicine, IGBMC, Illkirch, France FREDERIC BOCCARD • Centre de Génétique Moléculaire, CNRS, Yvette, France PHILIPPE BOUVET • Laboratoire de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, France RAYMOND S. BROWN • Laboratory of Molecular Medicine, Howard Hughes Medical Institute, Children’s Hospital, Boston, MA MALCOLM BUCKLE • Unité Physicochimie des Macromolécules Biologiques, Institut Pasteur, Paris, France MICHAEL J. BUMBULIS • Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, and the Department of Biology, Baldwin-Wallace College, Berea, OH STEPHEN BUSBY • School of Biochemistry, University of Birmingham, Birmingham, UK MARK L. CARPENTER • University of Oxford, Oxford, UK DAVID R. CHAFIN • Department of Biochemistry, University of Rochester, Rochester, NY YEN C HOO • Laboratory of Molecular Biology, Medical Research Council, Cambridge, UK RACHEL M. CONLIN • Laboratory of Molecular Medicine, Howard Hughes Medical Institute, Children’s Hospital, Boston, MA xiii

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BERNARD A. CONNOLLY • Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK BENOÎT COULOMBE • Départment de Biologie, Centre de Recherche sur les Méchanismes d’Expression Génétique, Université de Sherbrooke, Sherbrooke, Québec, Canada LUDMILLA DENISSOVA • Max Planck Institute of Biochemistry, Martinsried, Germany STEFAN I. DIMITROV • Faculté de Médecine, Institut Albert Bonniot, Université Joseph Fourier Grenoble I, La Tronche, France QIANPING DONG • Waksman Institute and Department of Chemistry, Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ KLAUS DOSE • Institut für Biochemie, Johannes Gutenberg-Universität, Mainz, Germany RÉGEN DROUIN • Department de Biologie Médicale, Université Laval, et Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche, Pavilion Saint-Francois d’Assise, Québec, Canada RICHARD H. EBRIGHT • Waksman Institute and Department of Chemistry, Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ LISA E. ENGLER • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA ALEXANDER A. GALL • Seattle Genetics, Bothell, WA JOHANNES GEISELMANN • Plasticité et Expression des Génomes Microbiens, Université Joseph Fourier, Grenoble, France CARLA W. GRAY • Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX SYLVAIN GUÉRIN • Laboratory of Molecular Endocrinologie, Centre Hopitalier Universitaire de Québec, Université Laval, Québec, Canada JEFFREY J. HAYES • Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY HERMANN HEUMANN • Max Planck Institute of Biochemistry, Martinsried, Germany MARK ISALAN • Laboratory of Molecular Biology, Medical Research Council, Cambridge, UK ILIAN JELESAROV • Biochemisches Institut der Universität Zurich, Zurich, Switzerland LINDA JEN-JACOBSON • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA BRENDA F. KAHL • Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO YOUNGGYU KIM • Waksman Institute and Department of Chemistry, Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ

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G. GEOFF KNEALE • Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK ANNIE KOLB • Institut Pasteur, Paris, France MICHAEL R. KURPIEWSKI • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA SIMON LABBÉ • Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, MI MARC-ANDRÉ LANIEL • Laboratory of Molecular Endocrinologie, Centre Hopitalier Universitaire de Québec, Université Laval, Québec, Canada OLIVIER LAROCHELLE • Centre de Recherche en Cancérologie, Université Laval, CHUQ/L´Hotel-Dieu de Québec, Québec, Canada LUC LEBEAU • Faculté de Médecine, Illkirch, France BENOIT LEBLANC • NIDDK, NIH, Bethesda, MD HSIAO-HUI LIU • Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK YURI L. LYUBCHENKO • Departments of Biology and Microbiology, Arizona State University, Tempe, AZ IAN W. MANFIELD • Department of Genetics, University of Leeds, Leeds, UK JAMES A. MCCLELLAN • Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK RAINER MEFFERT • Ministerium für Umwelt und Forsten des Landes RheinlandPfalz, Mainz, Germany WILLI METZGER • Ministerium für Umwelt und Forsten des Landes RheinlandPfalz, Mainz, Germany TOM MOSS • Centre de Recherche en Cancérologie et départment de Biologie Médicale de l’Université Laval, Centre Hopital Universitaire de Québec, Québec, Canada NIKOLAI NARYSHKIN • Waksman Institute and Department of Chemistry, Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ PETER E. NIELSEN • Department of Medical Biochemistry and Genetics, Laboratory of Biochemistry, The Panum Institute, Copenhagen, Denmark ANTHONY W. OLIVER • Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK STÉPHANE OUELLET • Department de Biologie Médicale, Université Laval, et Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche, Pavilion Saint-Francois d’Assise, Québec, Canada ATHANASIOS G. PAPAVASSILIOU • Department of Biochemistry, School of Medicine, University of Patras, Patras, Greece DAMIAN PARRY • Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK

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MARVIN PAULE • Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO JIM PERSINGER • Department of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University, Carbondale, IL SIMON E. PLYTE • Pharmacia and Upjohn, Milano, Italy GUY G. POIRIER • Unité Santé et Environment, CHUQ, Pavillon CHUL, Québec, Canada GABRIELE RATHGEBER • Merck KGaA, Darmstadt, Germany CHRISTOPHER M. READ • Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK VIRGIL RHODIUS • School of Biochemistry, University of Birmingham, Birmingham, UK FRANÇOIS ROBERT • Whitehead Institute for Biomedical Research, Cambridge, MA CHRISTOPH RUSSMANN • Fachbereich Physik, Universität Kaiserlautern, Germany NIGEL SAVERY • School of Biochemistry, University of Birmingham, Birmingham, UK HANS-JOCHEN SCHAFER • Institute für Biochemie, Johannes Gutenberg-Universität, Mainz, Germany PETER SCHICKOR • Max Planck Institute of Biochemistry, Martinsried, Germany DEBORAH B. SCHULMAN • Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH PATRICK SCHULTZ • Faculté de Médecine, Illkirch, France CARL SÉGUIN • Centre de Recherche en Cancérologie, Université Laval, CHUQ/L´Hotel-Dieu de Québec, Québec, Canada DAVID R. SETZER • Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH PETER E. SHAW • Department of Biochemistry, School of Biomedical Sciences, University of Nottingham, Queen’s Medical Center, Nottingham, UK LUDA S. SHLYAKHTENKO • Departments of Plant Biology and Microbiology, Arizona State University, Tempe, AZ MARTHA N. SIMON • Brookhaven National Laboratory, Biology Department, Upton, NY A. FRANCIS STEWART • European Molecular Biology Laboratory, Heidelberg, Germany GALE STEWART • Centre de Recherche en Cancérologie, Université Laval, CHUQ/L´Hotel-Dieu de Québec, Québec, Canada PETER G. STOCKLEY • Department of Genetics, University of Leeds, Leeds, UK IAN TAYLOR • Laboratory of Molecular Biophysics, University of Oxford, Oxford, UK

Contributors

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JEAN-PHILIPPE THERRIEN • Division de Pathologie, Department de Biologie Médicale, Université Laval, et Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche, Pavilion Saint-Francois d’Assise, Québec, Canada ANDREW A. TRAVERS • Lab Molecular Biology, Medical Research Council, Cambridge, UK JOSEPH S. WALL • Brookhaven National Laboratory, Biology Department, Upton, NY MICHELLE WEBB • Department of Chemistry, University of Sheffield, Sheffield UK EVGENY ZAYCHIKOV • Max Planck Institute of Biochemistry, Martinried, Germany CHRISTIAN ZWIEB • Department of Molecular Biology, The University of Texas Health Center at Tyler, Tyler, TX

Filter-Binding Assays

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1 Filter-Binding Assays Peter G. Stockley 1. Introduction Membrane filtration has a long history in the analysis of protein–nucleic acid complex formation, having first been used to examine RNA–protein interactions (1), before being introduced to DNA–protein interaction studies by Jones and Berg in 1966 (2). The principle of the technique is straightforward. Under a wide range of buffer conditions, nucleic acids pass freely through membrane filters, whereas proteins and their bound ligands are retained. Thus, if a particular protein binds to a specific DNA sequence, passage through the filter will result in retention of a fraction of the protein–DNA complex by virtue of the protein component of the complex. The amount of DNA retained can be determined by using radioactively labeled DNA to form the complex and then determining the amount of radioactivity retained on the filter by scintillation counting. The technique can be used to analyze both binding equilibria and kinetic behavior, and if the DNA samples retained on the filter and in the filtrate are recovered for further processing, the details of the specific binding site can be probed by interference techniques. The technique has a number of advantages over footprinting and gel retardation assays, although there are also some relative disadvantages, especially where multiple proteins are binding to the same DNA molecule. However, filter binding is extremely rapid, reproducible, and, in principle, can be used to extract accurate equilibrium and rate constants (3–5). We have used the technique to examine the interaction between the E. coli methionine repressor, MetJ, and various operator sites cloned into restriction fragments (6,7, see also Chapter 15). Results from these studies will be used to illustrate the basic technique.

From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Before discussing the experimental protocols it is important to understand some fundamental properties of the filter-binding assay. The molecular basis of the discrimination between nucleic acids and proteins during filtration is still not fully understood. Care should therefore be taken to characterize the assay with the system under study. Nucleic acid–protein complex retention occurs with differing efficiencies, depending on the lifetime of the complex, the size of the protein component, the buffer conditions, and the extent of washing of the filter. Experiments with the lac repressor system have shown that prior filtration of protein followed by passage of DNA containing operator sites does not result in significant retention of the nucleic acid, presumably because filter-bound protein is inactive for further operator binding. The DNA retained on filters is therefore a direct reflection of the amount of complex present when filtration began. Furthermore, incubation of the lac repressor with large amounts of DNA that does not contain an operator site followed by filtration also does not lead to significant retention. Because the lac repressor (and, indeed, essentially all DNA-binding proteins) binds nonsequence-specifically to DNA, forming short-lived complexes, it is clear that these are not readily retained. The experiments with the lac repressor (3–5) can therefore be used as a guide when designing experimental protocols. The repressor is a large protein (being a tetramer of 38-kDa subunits) but the basic features seem to apply even to short peptides with molecular weights 95%. An example of the sort of results obtained with the MetJ repressor is shown in Fig. 1. 2. Materials 2.1. Preparation of Radioactively End-Labeled DNA 1. Plasmid DNA carrying the binding site for a DNA-binding protein on a convenient restriction fragment (usually 6000 Ci/mmol, and the other three dNTPs as nonradioactive 2 mM stocks. Agarose gel with slots large enough to take >60 mL (they can be made by taping up smaller slots), powerpack and gel tank. 5X loading dye: 10% Ficoll 400, 0.1 M sodium EDTA, 1% sodium dodecyl sulfate (SDS), 0.25% bromophenol blue, and 0.25% xylene cyanol. Ethidium bromide (10 mg/mL in water). Long-wavelength (360-nm) ultraviolet (UV) transilluminator. Electroeluter. We use the IBI model UEA. 1 M aqueous piperidine, made freshly. High-capacity vacuum pump with trap and desiccator. Alkaline formamide dye; 200 µL deionized formamide, 1 µL of 1 M NaOH and xylene cyanol and bromophenol blue to taste. (A needle dipped in the powdered dye and then tapped to remove excess will give quite enough.) Whatmann 3MM paper.

3. Methods 3.1. Osmium Tetroxide Modification This protocol describes the in vitro modification of plasmid DNA and the sequence-level detection of adducts by piperidine cleavage after specific 3-prime end labeling. 1. Solution 1: Combine the plasmid DNA with 5 µL of 10X modification buffer and add distilled water to a total volume of 45 µL. 2. Solution 2: Mix 2.5 µL of 20 mM osmium tetroxide with 2.5 µL of 6% pyridine or 20 mM bipyridine. 3. Equilibrate solutions 1 and 2 at the desired reaction temperature. 4. Add solution 1 to solution 2, mix well, and incubate for the desired time. 5. Meanwhile, make the stop solution; 180 mL of absolute ethanol and 5 mL of 3 M sodium acetate pH 4.5. Chill at –70°C. 6. Add stop solution to reaction, mix well and chill at –70°C for 10 min. 7. Spin at maximum speed in a Eppendorf microfuge for 10 min. Note, nonEppendorf microcentrifuges are NOT, in general, adequate substitutes. We do not know exactly why, but we suspect it has to do with heating of the rotor during spinning. 8. Pipet off the supernatant and discard as potentially carcinogenic waste. Be careful not to disturb the pellet. 9. Add 1 mL of absolute ethanol to the tube and spin in the microfuge at maximum speed for 5 min. 10. Pipet off the supernatant and discard as in step 8. 11. Dry the pellet for 5 min in a vacuum desiccator. 12. Add 44 µL of distilled water. 13. Transfer to a fresh tube. This is important; sometimes residual osmium on the reaction tube can interfere with the subsequent steps (see Note 4).

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14. Add 5 µL of 10X restriction enzyme buffer and 1 µL (about 10 U) of restriction enzyme. The enzyme chosen should give unpaired 5' ends and ideally has a unique site approx 40–200 bases from the site at which modification is expected. 15. Incubate at 37°C for about 1 h. 16. Meanwhile, dry down 5 mCi of a >6000-Ci/mmol [α–32P] dNTP. This should be the first nucleotide to be incorporated by DNA polymerase (e.g., if the enzyme used is EcoRI, it should be dATP). 17. To the dried-down radionucleotide, add 2 µL of each 2 mM unlabeled stocks of the other dNTPs (i.e., if the labeling is with [α–32P] dATP, add dGTP, dCTP, and dTTP). Mix well. 18. Add this nucleotide mix to the restriction digest. Then, add 1 µL (about 6 U) of the Klenow fragment of E. coli DNA polymerase I. We find that Klenow polymerization works well in a variety of restriction enzyme buffers, especially those used for EcoRI and BamHI. 19. Incubate for 1 h (not more) at 37°C. 20. Add 10 µL of 5X loading dye and electrophorese on, for example, a 1% agarose 1X TBE gel until the xylene cyanol (light blue) dye is about halfway down the gel, or, in general until the fragment of interest can be easily excised from the gel. Exactly which gel is chosen will depend on the particular system under study. 21. Stain the gel with 1 µg/mL ethidium bromide. Visualize by long-wave UV and excise the bands of interest from the gel. 22. Electrolelute the labeled DNA into high salt (or use another method). 23. Precipitate and wash the DNA with ethanol. If precipitating from high salt, do not chill! 24. If it is desired to see only the signals on one strand, cut off one of the labeled ends (Note 5). After restriction, the DNA should be ethanol precipitated, washed and dried. Do this by repeating steps 5–11; it is not necessary to discard the waste as potentially mutagenic. 25. To the dried pellet, add 100 µL of 1 M piperidine. Heat at 90°C for 30 min. 26. Transfer to a new tube. Close the tube. Use a needle to punch holes in the cap. 27. Place the tube in liquid nitrogen for about 5 s. 28. Place the tube in a rack in a vacuum desiccator, attach a high-capacity vacuum pump with a trap, and turn it on. Do this quickly, so that the frozen sample does not have time to thaw. 29. Lyophilize for about 2 h. 30. Add 50 µL of water to the tube and repeat lyophilization step 28 for about 1 h. 31. Repeat steps 29 and 30. 32. Resuspend the samples in at least 3 µL of alkaline formamide dye. 33. Heat to 90°C for 5 min and then chill on ice. 34. Load aliquots of about 50 counts per second on a standard TBE–8 M urea sequencing gel and electrophorese. Exact details of the sequencing gel will depend on the system under study. 35. Fix the gel in 10% acetic acid for 15 min, transfer to 3MM paper, dry, and autoradiograph.

Osmium modification 125 3.2. Results of Osmium Tetroxide Modification Figure 1 shows a time-course of osmium tetroxide modification on a 68-bplong tract of alternating adenines and thymidines within a bacterial plasmid. This sequence is found in the first intron of the frog globin gene. The modification was done at 37°C in the absence of added salt and for the indicated times. A BamHI–EcoRI fragment containing the tract was labeled at both ends by Klenow polymerase and [α-32P] dATP. The adducts were cleaved using hot piperidine and electrophoresed on a sequencing gel. The gel was fixed, dried onto paper, and autoradiographed. The gel shows information from both strands. This is possible because (1) the AT tract is asymmetrically placed on the BamHI–EcoRI fragment, and (2) osmium modification is almost completely specific for the AT tract. As can be seen, the modifications are biased toward the label-proximal end of the tract on both strands, and this is intensified at later time-points. In fact, this is also the pattern when the label is placed at the 5' ends (data not shown). Thus, the label-proximal bias of the signals simply indicates multiple modifications rather than an asymmetric structure. Figure 1 also shows the result of modifying the AT tract in the presence of ions. Here, the patterns are quite different. In the presence of sodium ions, we see modifications at the center of the AT tract and at its ends. In the presence of magnesium ions, we see modifications at the center of the AT tract but not at its ends. These patterns are interpreted as indicating the presence of cruciform structures at the AT tract, either with tight scissor-shaped osmium resistant junctions (in the presence of magnesium) or with floppy square planar osmium sensitive junctions (in the presence of sodium) (16). The central modifications are at what would be the loop of the cruciform, and the modifications at the end of the tract would be at the junction of the cruciform with flanking DNA (Fig. 2) (see Note 6). The nature of the symmetric structure observed in Fig. 1 remains obscure; we have termed it the U-structure (9), but this is only a name, not a description. What we know about this structure is that it is easy to interconvert between it and the cruciform, and the conditions under which the U-structure is favored (higher supercoiling and temperature, lower salt concentration) suggest that the U-structure is more unwound than the cruciform. Furthermore, other A+Trich sequences that form cruciforms can exhibit U-like patterns of modification under the appropriate conditions. One interesting possibility is that the U-structure may be a locally parallel-stranded conformation, in which the A at the 5' end of the top strand makes a reverse Watson–Crick pair to the A at the 5' end of the bottom strand; the Ts following those As pair to each other in the same mode, the As following those Ts pair to each other in the same mode: and

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Fig. 1. Time-course of in vitro osmium tetroxide modification of (AT)34 tract in plasmid pXG540. The figure shows an autoradiograph of end-labeled piperidinecleaved EcoRI–BamHI fragments from plasmid pXG540 that had been treated with osmium tetroxide under the ambient conditions indicated above each lane (all reactions were done at 20°C). The fragments were separated on a 6% sequencing gel run hot to the touch at constant 70 W.

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Fig. 2. Two conformations of cruciforms. The figure shows how two different cruciform conformations may be postulated in order to explain the different patterns of osmium modification seen at different ionic strengths.

so on. This is chemically plausible because any base can make a reverse Watson-Crick pair with two hydrogen bonds to another base of the same type (i.e., A pairs to A, T to T, and so on). The proposed structure is shown diagrammatically in Fig. 3, and the model is currently being tested.

3.3. In Vivo Osmium Modification It is possible to modify AT tracts in plasmids inside living bacteria (10–12). Figure 4 shows the results of one such experiment, in which a number of plasmids with different lengths of AT were modified inside bacterial cells (E. coli HB101). The plasmid DNA was recovered by a modification of the Holmes– Quigley boiling method (12); alkaline lysis methods are not used, in order to avoid premature alkaline cleavage of adducts. The DNA was then restricted and end labeled, and the adducts were cleaved by hot piperidine before analysis on sequencing gels, as described earlier. In vivo modification requires a number of additional tricks if it is to be successful. One has to use bipyridine as the ligand. The number of cells is a critical parameter; the modification reaction should have cells at an optical density (OD) 550-nm of 0.4. If the OD is even twice as high, the experiment is likely to fail. During the modification, the cells should turn a milk chocolate brown; if they do not, discard the experiment and obtain fresh osmium tetroxide. We

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Fig. 3. Proposed locally parallel structure of U-conformation.

usually work on a 50-mL culture scale, and we usually wash the cells and do the modifications in 100 mM potassium phosphate buffer, pH 7.4. It is, however, possible to do the modifications in L-broth. After the cells have been boiled the supernatant has to be collected, and it is very viscous indeed. We find that the best way to prepare the supernatant is to spin the lysates at 30,000 rpm for 30 min in a Beckmann table-top ultracentrifuge (120 TS rotor). The supernatant can then be ethanol precipitated or made up as a CsCl/ethidium bromide gradient. 4. Notes 1. Osmium tetroxide is a very powerful oxidizing and crosslinking reagent, which formerly was used for tanning leather. It can react explosively with water. Fumes of osmium tetroxide can damage the cornea of the eye. It is, thus, a chemical to be treated with great respect. To our knowledge, there is no evidence that it is a carcinogen, but this may be because the complex with heterocyclic activators has not been tested as such; this complex is certainly a very powerful and specific covalent modifier of exposed thymidines, and it would be surprising if it was not a carcinogen. It therefore seems prudent to dispose of osmium tetroxide waste as if it were carcinogenic. 2. Osmium tetroxide can be bought from various suppliers, including Sigma and Johnson Matthey. The quality of the reagent varies widely, but, in general, it may be stated that the material bought from Johnson Matthey is superior. One usually buys a 250-mg aliquot, which should cost around £30–40 sterling at 1998 prices. The reagent is shipped inside a metal can. This contains a plastic tube, and inside the tube, wrapped in black paper, is a little sealed glass phial containing yellow crystals. These are osmium tetroxide. One makes up a stock 20 mM solution as follows: a. The glass tube is plunged into liquid nitrogen and kept there for about 15 s. This treatment prevents the chemical from reacting explosively with water, and also it makes the crystals less sticky and easier to remove from the glass vial. b. Inside the fume hood and over a washable tray, the glass vial is broken, ideally with the aid of a diamond knife, and the crystals are tipped into a glass beaker containing 49.2 mL of distilled water. The crystals take some time to dissolve, about 2–3 h.

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Fig. 4. In vivo modification of AT tracts: cruciform geometry in bacterial plasmids as a consequence of physiological salt shock. Bacterial cells containing isogenic plasmids with various lengths of AT were salt-shocked and treated with osmium tetroxide in vivo. Adducts were detected by preparing the DNA, end labeling, and piperidine cleavage, followed by electrophoresis on thin 6% sequencing gels and autoradiography. Strong central modification of the AT tract shows that (AT)34 adopts cruciform geometry in salt-shocked but not in control cells, and that (AT)22 and (AT)15 but not (AT)12 also adopt cruciform geometry in salt-shocked cells. c. Once the crystals have dissolved, the reagent is aliquoted ready for use. Glass containers such as Universals with screw tops should be used, and storage should be at –70°C. It is best to keep the reagent as several separate aliquots

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Fig. 5. Stereochemistry of OsO4 attack on thymidines. and to use them one at a time. One problem with storage is that the glass containers often crack, which is obviously very dangerous. Volumetric flasks and non-Pyrex containers are particularly sensitive in this regard. The reagent should always be thawed with the container inside a glass beaker in the fume hood. If the reagent is blackish when thawed, discard it; the black color indicates lower oxidation states of osmium, including the metal. 3. By itself, osmium tetroxide is not very reactive with DNA. The species that attacks DNA is a complex of osmium tetroxide with a heterocyclic compound such as pyridine or bipyridine (Fig. 5). The attack is on the 5–6 double bond of thymidines (ref. 3 and references therein). Curiously, neither cytosines nor uracils show significant modifiability with osmium tetroxide, but guanine residues occasionally may. Bipyridine is rather insoluble in water. To make a stock solution, it may be necessary to heat the bipyridine and water to 80°C for about 30 min. Once osmium tetroxide is added to pyridine, the mixed reagents should become a strawyellow color; if they do not, discard them and obtain fresh osmium tetroxide. One can change the reactivity of an osmium tetroxide preparation by changing the concentration of osmium and/or ligand, and the overall degree of reaction can be varied from single to multiple hits per molecule by changing the time and/or temperature of the modification reaction. Typical times and temperatures used in our laboratory to obtain single hits are as follows: 45 min at ice temperature; 15 min at 20°C; 5 min at 37°C; 1 min at 40°C. 4. Various methods of detecting osmium tetroxide adducts exist, namely retardation of bands in agarose or acrylamide gels (5), immunoprecipitation (3), cleavage by S1 nuclease (5), cleavage by hot piperidine (6), and failure of primer extension (11). In some cases, inhibition of restriction enzyme cleavage can be used (10,17).

Osmium modification 131 5. A good way to do this is to cut with a frequent cutter that a. has a site near to the end from which it is not required to see signals b. does not have a site between the end from which it is desired to see signals and the tract where modifications are expected For example, the EcoRI site of pXG540 has a HaeIII site 18 bp anticlockwise (in the direction of the Amp promoter). There is no HaeIII site in the clockwise 170 bp up to the start of the AT tract. So, we often cut and label at EcoRI, and then do a limit digest with HaeIII. This reults in a long labeled fragment that has the information we want on it, and a short fragment (18 bp) that we run off the gel. 6. Osmium tetroxide modification of DNA now has a substantial pedigree, having been used to modify a wide range of sequences in different unusual conformations in vitro, and a narrower range of sequences in different conformations in vivo. Figure 6 shows some of the actual or proposed structures that have been treated with osmium tetroxide. However, it is very important to be cautious in using osmium modification results, or any other kind of chemical or enzymatic probing, to deduce that a particular structure is forming. This is because osmium does not report on global conformation; it only tells you whether or not a particular T residue has an exposed 5–6 double bond. If a T is exposed, this could be for several reasons: a. The T might be in a single-stranded region of the DNA (e.g., a bubble, mismatch [18], B–Z junction or cruciform or H-structure loop). b. The T might be in a helix with a shallow or bulging major groove (e.g., at a bend or within a GT/AC tract that was forming Z–DNA). c. The T might be in an overwound helix, with exposure of the 5–6 double bond resulting from a loss of base overlap, because of the sharp rotation of each base relative to its neighbors. These considerations make it very difficult to conclude on the basis of osmium modification that, for example, Z–DNA is forming; osmium results do not distinguish in any simple way between what we have called the “U”-structure (9), Z-DNA and a conformation (possibly the eightfold D-helix), which we have observed in locally positively stressed AT tracts (19). Detecting the loops and junctions of cruciform or H-form DNA can sometimes be done in such a way as to virtually exclude alternative interpretations of the data, but even this is not always possible; for example, a GC tract with ATAT in the middle will react in the same way with osmium tetroxide whether it is in a Z-conformation or cruciform (11); and there is a published interpretation of chemical modification at H-forming sequences that is radically at odds with the H-structure model (20). In addition to these difficulties, there is a need to be cautious about the effect of the probing chemical on the structural features deduced. Because osmium modification works under a wide range of conditions, it presents fewer such problems of interpretation than some other more fastidious chemicals. Nevertheless, we observe that the ligand used can have an effect on the result obtained; other things being equal, we find that bipyridine is more likely to give a cruciformlike

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Fig. 6. Unusual DNA structures that react with osmium tetroxide. pattern of modification and pyridine is more likely to give a U-structure-like pattern when superhelically stressed AT tracts are probed in vitro. This probably has to do with different helix-unstacking potential of the two heterocycles; alternatively, it could be an artifact of contaminating cations in the heterocycles or ion–heterocycle interactions.

Osmium modification 133 References 1. Beer, M., Stern, S., Carmalt, D., and Mohlenrich, K. H. (1966) Determination of base sequence in nucleic acids with the electron microscope. V. The thyminespecific reactions of osmium tetroxide with deoxyribonucleic acid and its components. Biochemistry 5, 2283–2288. 2. Burton, K. and Riley, W. T. (1966) Selective degradation of thymidine and thymine deoxynucleotides. Biochem. J. 98, 70–77. 3. Palecek, E. (1989) Local open DNA structures in vitro and in the cell as detected by chemical probes, in Highlights of Modern Biochemistry, (Kotyk, A., Skoda, J., Paces, V., and Kostka, V., eds.), VSP International Science, Zeist, The Netherlands, pp. 53–71. 4. Behrman, E. J. (1988) The chemistry of the interactions of osmium tetroxide with DNA and proteins, in Symposium on Local Changes in DNA Structure and Their Biological Implications, Book of Abstracts, p. 6. 5. Buckle, M., Spassky, A., Herbert, M., Lilley, D. M. J., and Buc, H. (1988) Chemical probing of single stranded regions of DNA formed in complexes between RNA polymerase and promoters, in Symposium on Local Changes in DNA Structure and their Biological Implications, Book of Abstracts, p. 11. 6. Lilley, D. M. J. and Palecek, E. (1984) The supercoil-stabilised cruciform of ColE1 is hyper-reactive to osmium tetroxide. EMBO J. 3, 1187–1192. 7. Johnston, H. and Rich, A. (1985) Chemical probes of DNA conformation: detection at nucleotide resolution. Cell 42, 713–724. 8. Vojtiskova, M. and Palecek, E. (1987) Unusual protonated structure in the homopurine. homopyrimidine tract of supercoiled and linearised plasmids recognised by chemical probes. J. Biomol. Struct. Dyn. 5, 283–296. 9. McClellan, J. A. and Lilley, D. M. J. (1987) A two-state conformational equilibrium for alternating A-T)n sequences in negatively supercoiled DNA. J. Mol. Biol. 197, 707–721. 10. Palecek, E., Boublikova, P., and Karlovsky, P. (1987) Osmium tetroxide recognizes structural distortions at junctions between right- and left-handed DNA in a bacterial cell. Gen. Physiol. Biophys. 6, 593–608. 11. Rahmouni, A. R. and Wells, R. D. (1989) Stabilisation of Z DNA in vivo by localized supercoiling. Science 246, 358–363. 12. McClellan, J. A., Boublikova, P., Palecek, E., and Lilley, D. M. J. (1990) Superhelical torsion in cellular DNA responds directly to environmental and genetic factors. Proc. Natl. Acad. Sci. USA 87, 8373–8377. 17. Palecek, E., Boublikova, P., Galazka, G., and Klysik, J. (1987) Inhibition of restriction endonuclease cleavage due to site-specific chemical modification of the B–Z junction in supercoiled DNA. Gen. Physiol. Biophys. 6, 327–341. 16. Duckett, D. R., Murchie, A. I. H., Diekmann, S., von Kitzing, E., Kemper B., and Lilley, D. M. J. (1988) The structure of the Holliday junction, and its resolution. Cell 55, 79–89. 13. Dorman, C. J. (1991) DNA supercoiling and environmental regulation of gene expression in pathogenic bacteria. Inf. Immun. 59, 745–749.

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14. Langermann, S. and Wright, A. (1990) Molecular analysis of the Haemophilus influenzae type b pilin gene. Mol. Microbiol. 4, 221–230. 15. Horwitz, M. S. Z. and Loeb, L. A. (1988) An E. coli promoter that regulates transcription by DNA superhelix-induced cruciform extrusion. Science 241, 703–705. 18. Cotton, R. G. H., Rodrigues, N. R., and Campbell, R. D. (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its app. lication to the study of mutations. Proc. Natl. Acad. Sci. USA 85, 4397–4401. 19. McClellan, J. A. and Lilley, D. M. J. (1991) Structural alteration in alternating adenine–thymine sequences in positively supercoiled DNA. J. Mol. Biol. 219, 145–149. 20. Pulleyblank, D. E., Haniford, D. B., and Morgan, A. R. (1985) A structural basis for S1 sensitivity of double stranded DNA. Cell 42, 271–280.

Combined SW–Nuclease Footprinting

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10 Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots Athanasios G. Papavassiliou 1. Introduction The interaction of cell-type-specific or inducible transcription factors with regulatory DNA sequences in gene promoters or enhancers is a pivotal step in genetic reprograming during cell proliferation and differentiation and in response to extracellular stimuli. The study of these interactions and the characterization of the factors involved are, therefore, a critical aspect of gene control. Transcription factor–DNA interactions in eukaryotes have been demonstrated by a wide variety of biochemical approaches, including deoxyribonuclease I (DNase I) and chemical nuclease footprinting (1–3) (Chapter 3), methylation protection (4) (Chapter 14), electrophoretic mobility-shift (5,6) (Chapter 2), and Southwestern (SW) assays (7) (Chapter 17). Despite their broad applicability, these techniques provide only partial information about the DNA–protein system under investigation. The first three techniques identify either the site(s) of transcription factor binding within the DNA (size and location of nucleotide stretches or atoms on individual bases) or the complexity of the binding pattern (stoichiometry), but do not yield information about the protein(s) involved. On the other hand, the SW assay reveals the relative molecular mass of renaturable (on a membrane support) active species in heterogeneous protein mixtures facilitating their identification, but fails to localize the exact target element within the probing DNA sequence. Combining SW and DNase I (but also chemical nuclease and methylation protection, see below) footprinting methodologies has the dual potential for accurately determining the size of individual DNA-binding transcription factors and precisely mapping their cognate binding sites (8) (Fig. 1) . In addition From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Fig. 1. Rationale in a combined SW–DNase I footprinting procedure. The “hammer”-shaped extensions from the DNA-binding protein indicate immobilization on the blotting membrane. The black-filled sphere indicates the end label in one strand of the DNA footprint probe. MW denotes the molecular size of the membrane-blotted DNA-binding protein (estimated by comparing the position of the active membrane area to the mobilities of coelectroblotted protein MW standards). Arrows mark representative sites of DNase I attack. (A) DNase I digestion pattern of free (in solution) probe; (B) DNase I digestion pattern of protein-complexed (membrane-bound) probe.

to allowing the detection of only fractional binding (footprints in solution can be obtained only when the binding site[s] is almost completely occupied), coupling in situ DNase I footprinting with the SW technique offers the advantage of both resolving the protein component and mapping the binding site of complexes formed by either different factors recognizing an identical sequence within the DNA probe or by two factors interacting in a noncooperative manner with adjacent but distinct sequences. However, this is dependent on the factor being able to bind to DNA as a monomer or a homodimer. If the active form is not a single species (i.e., a heterodimeric or heteromeric complex is required to reconstitute the binding activity), specific DNA binding will not be detected and the procedure will not be applicable. The most critical stage of the coupled assay lies within its first part, namely the SW procedure, and concerns the ability of a transcription factor to efficiently renature into its active form (at least in terms of DNA-binding capacity) on the membrane. Many transcription factors are composed of domains with distinct structural conformations, which aids the process of renaturation. However, because the protein surface immobilized on the membrane poses an impediment on the refolding process, the


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likelihood of successful renaturation increases with increased size of the DNAbinding transcription factor(s) under study. As a result of the increased kinetic stability of a membrane-immobilized DNA–protein complex (reversible binding to even low-affinity proteins is enhanced because excess unbound DNA has been washed out and hence is not present to compete), reaction parameters such as the size of the DNA probe, the concentration of DNase I, and digestion time are no longer determined by the dissociation rate of the complex, a normal limitation of DNase I protection assays performed in solution. The fidelity of the combined analytical assay is demonstrated in Fig. 2. Evidently, the structural and functional properties of DNA are not altered by entrapment on the blotting membrane surface (i.e., the probe exhibits identical protection pattern and sequence-dependent reactivity with DNase I as it does in solution). Therefore, this coupled assay provides a fast and reliable method that will allow the user who has identified specific regulatory regions in the gene of interest to begin characterizing in detail the transcription factor(s) that bind to them in a certain cellular milieu. Modifications of the presented DNase I protection analysis on Southwestern blotting membranes (in situ) substitute the enzymatic probe for either the chemical nuclease 1,10-phenanthroline–copper ion (OP–Cu) (9) or dimethyl sulfate (DMS) (10). Both protocols have the additional advantage of providing information on the nature (i.e., specific–protected versus nonspecific– nonprotected) of several membrane-immobilized DNA–protein species often observed in a SW assay. The molecular and functional properties of DNase I (i.e., its relatively large size, mode of target searching and binding to cleave, and requirement for Mg2+ [which often stabilizes both specific and nonspecific complexes]) highly reduce its potential to detect these differences. In addition, these methodologies are useful in rapidly confirming the binding specificity of a protein isolated by screening a cDNA expression library with recognitionsite DNA. 2. Materials 2.1. SW Blotting

2.1.1. Solutions 1. Sodium dodecyl sulfate (SDS; 20%; w/v): Dissolve 100 g of SDS (Sigma, St. Louis, MO) in distilled, deionized water to a 0.5 L final volume; heat at 68°C to assist dissolution (do not autoclave). Store at room temperature in a clear bottle. A respirator or dust mask should be worn when handling powdered SDS. 2. Lower (separating) gel buffer (4X stock): 1.5 M Tris-HCl, pH 8.8, and 0.4% (w/v) SDS. Filter through a 0.22-µm membrane filter. Store at 4°C. 3. Upper (stacking) gel buffer (4X stock): 0.5 M Tris-HCl, pH 6.8, and 0.4% (w/v) SDS. Filter through a 0.22-µm membrane filter. Store at 4°C.

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Fig. 2. (A) Schematic outline of the manipulations involved in the combined SW–DNase I footprinting procedure. In the example presented, a crude lysate of bacterial cells overexpressing the proto-oncoprotein c-Jun (a component of the transcription factor AP-1) was subjected to a quantitative SW assay utilizing as probe a 134-bp DNA restriction fragment (5'-end labeled in the coding strand) encompassing the AP-1binding sequence of the human collagenase promoter [5'(-72)TGAGTCA3'(–66)]. (B) DNase I footprinting reactions of the same fragment in solution performed with increasing amounts (lanes 2–4) of the c-Jun preparation (lane 1, free-DNA probe). The footprinted region (solid bar) includes in both cases approx 14 bases centered around the AP-1-binding motif (20).

4. Acrylamide/bis-acrylamide gel mixture: 30% (w/v) acrylamide, 0.8% (w/v) N,N'methylene–bis-acrylamide (Bio-Rad, Richmond, CA). Filter through a 0.22-µm membrane filter. Store at 4°C in the dark. Powdered acrylamide and N,N'-methylene-bis-acrylamide are potent neurotoxins and are absorbed through the skin. Their effects are cumulative. Wear gloves and a face mask when weighing these substances and when handling solutions containing them (work in a chemical fume hood). Although polyacrylamide is considered to be nontoxic, it should be handled with care because of the possibility that it might contain small quantities of unpolymerized acrylamide.


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5. Ammonium persulfate (10%; w/v): Dissolve (by vigorous vortexing) 1 g of ammonium persulfate (Bio-Rad) in 10 mL of distilled, deionized water. Filter through a 0.22-µm membrane filter. Store at 4°C; make fresh weekly. Ammonium persulfate is extremely destructive to tissue of the mucous membranes and upper respiratory tract, eyes, and skin. Inhalation may be fatal. Exposure can cause gastrointestinal disturbances and dermatitis. Wear gloves, safety glasses, respirator, and other protective clothing and work in a chemical fume hood. Wash thoroughly after handling. 6. SDS–polyacrylamide gel electrophoresis (PAGE) running buffer (10X stock): 0.25 M Tris base (Sigma) and 1.92 M glycine (Sigma). It is not necessary to adjust the pH. The proportions of Tris base and glycine give pH 8.3. Store at room temperature in a large vessel (carboy). Dilute to 1X with distilled, deionized water, then add SDS to a final concentration of 0.1% (w/v). 7. SDS-PAGE sample buffer (4X stock): 0.5 M Tris–HCl pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol (Sigma), and 0.4% (w/v) bromophenol blue (Sigma). Store in aliquots at –20°C. Before use, make a working solution of SDS-PAGE sample buffer by diluting the 4X stock buffer and adding 2-mercaptoethanol (Sigma) to a final concentration of 5% (v/v) (see Note 1). 2-Mercaptoethanol is harmful if inhaled or absorbed through the skin. High concentrations are extremely destructive to the mucous membranes, upper respiratory tract, skin, and eyes. Use only in a chemical fume hood. Gloves, safety glasses, and respirator should be worn. 8. Western transfer buffer: 50 mM Tris base, 380 mM glycine, 0.1% (w/v) SDS, and 20% (v/v) methanol (see Note 2). There is no need to adjust the pH of this buffer by the addition of acid or alkali. Store at room temperature in a large vessel. 9. Dithiothreitol (stock): Prepare a stock solution of 0.5 M dithiothreitol (Sigma) in distilled, deionized water and store in aliquots at –20°C. (See safety note in item 7.) 10. Phenylmethylsulfonyl fluoride (PMSF; stock): Prepare a 100 mM stock solution of PMSF (Boehringer, Indianapolis, IN) in absolute ethanol and store in aliquots at –20°C. PMSF is extremely destructive to the mucous membranes of the respiratory tract, the eyes, and the skin. It may be fatal if inhaled, swallowed, or absorbed through the skin. It is a highly toxic cholinesterase inhibitor. Therefore, it should be used in a chemical fume hood and gloves and safety glasses should be worn during handling. 11. 1 M KCl. 12. 0.5 M MgCl2. 13. SW blocking/renaturation buffer: 3% (w/v) nonfat dried milk (or 5% [w/v] lipidfree bovine serum albumin [BSA]; see Note 3a), 25 mM HEPES·KOH, pH 7.5, 50 mM KCl, 6.25 mM MgCl2, 1 mM dithiothreitol (freshly added from the 0.5 M stock solution immediately before use), 10% (v/v) glycerol, 0.1% (v/v) Nonidet P-40 (BDH, London, UK), and 0.2 mM PMSF (freshly added from the 100 mM stock solution just prior to use). Store at 4°C. Prepare also some SW blocking/ renaturation buffer minus dried milk (or BSA).

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14. Poly(vinyl alcohol) (stock): Prepare a 10% (w/v) stock solution of poly(vinyl alcohol) (Sigma P 8136; average mol. wt. = 10,000) in distilled, deionized water and store at –20°C. 15. SW binding/washing buffer: 12.5 mM HEPES.KOH, pH 7.5, 50 mM KCl, 6.25 mM MgCl2, 0.5 mM dithiothreitol (freshly added from the 0.5 M stock solution immediately before use), 2% (w/v) polyvinyl alcohol, 10% (v/v) glycerol, 0.05% (v/v) Nonidet P-40, and 0.2 mM PMSF (freshly added from the 100 mM stock solution just prior to use). Store at 4°C. (See Note 4b.) 16. Phenol/chloroform/isoamyl alcohol (25:24:1; v/v): Mix just prior to use 25 vol of phenol with 24 vol of chloroform and 1 vol of isoamyl alcohol. Phenol is highly corrosive and can cause severe burns. Any areas of skin that come in contact with phenol should be rinsed with a large volume of water or PEG 400 (Sigma) and washed with soap and water (do not use ethanol!). Chloroform is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is also a carcinogen and may damage the liver and kidneys. Wear gloves, protective clothing, safety glasses, and respirator when handling these substances and carry out all manipulations in a chemical fume hood. 17. Chloroform/isoamyl alcohol (24:1; v/v): Mix 24 vol of chloroform with 1 vol of isoamyl alcohol. This organic mixture can be stored at room temperature in dark (brown) bottles indefinitely. 18. Nonspecific competitor DNA (stock solution): Dissolve salmon/herring sperm or calf thymus DNA in distilled water, deproteinize it by sequential phenol/chloroform/isoamyl alcohol (25:24:1; v/v) and chloroform/isoamylalcohol (24:1; v/v) extractions, sonicate to reduce the mean DNA length to 100–200 bp, precipitate the DNA with ethanol, then resuspend it at 1 mg/mL in distilled, deionized water.

2.1.2. Reagents/Special Equipment 1. N,N,N',N'-tetramethylethylenediamine (TEMED; Bio-Rad). TEMED is extremely destructive to tissue of the mucous membranes and upper respiratory tract, eyes, and skin. Inhalation may be fatal. Prolonged contact can cause severe irritation or burns. Wear gloves, safety glasses, respirator, and other protective clothing and work in a chemical fume hood (TEMED is flammable!). Wash thoroughly after handling. 2. Protein extract of interest (e.g., whole-cell-free extract, crude nuclear extract, or partially purified extract), as concentrated as possible. 3. Prestained nonradioactive MW standards (Bio-Rad) or [14C]-methylated protein MW markers (Amersham). 4. Nonfat dried milk powder (e.g., Cadbury’s Marvel or Carnation), or fatty acidfree BSA (Boehringer; fraction V). 5. Phenol: Redistilled (under nitrogen) phenol equilibrated with 100 mM Tris-HCl, pH 8.0, and 1 mM EDTA in the presence of 0.1% (w/v) 8-hydroxyquinoline. Phenol can be stored at 4°C in dark (brown) bottles for up to 2 mo. See the relevant safety note in Subheading 2.1.1. 6. Absolute ethanol.


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7. Salmon/herring sperm or calf thymus DNA (Sigma or Boehringer). 8. Singly 32P end-labeled DNA probe bearing the binding site(s) of interest (see Note 5). All necessary precautions should be observed to minimize exposure to ionizing radiation during labeling and isolation of the probe; work behind protective screens whenever possible. 9. Radioactive ink: Mix a small amount of 32P with waterproof black drawing ink, to a concentration of approx 200 cps (on a Geiger counter) per microliter. 10. 0.22-µm membrane filters (Millipore, Bedford, MA). 11. Mini-slab gel electrophoresis apparatus, giving 0.5- to 1.0-mm-thick mini-gels (e.g., Bio-Rad Mini Protean II system), and accompanying equipment. 12. High-current (2–3 A) power supply (e.g., Bio-Rad or Hoefer, San Francisco, CA) and electroblotting apparatus for Western transfer (e.g., Bio-Rad Trans-Blot); additional equipment for Western transfer (11). 13. Nitrocellulose membrane: suitable membranes comprised of unsupported or supported nitrocellulose are available from a number of manufacturers, such as Schleicher & Schuell (Keene, NH; BA85, 0.45 µm), Millipore (Immobilon-NC), and Amersham (Hybond-C/C extra). 14. Plastic trays. 15. Forceps. 16. Plastic wrap such as cling film or Saran Wrap®. 17. X-ray film (e.g., Kodak X-Omat AR, Rochester, NY). 18. Additional equipment: protective gloves/glasses/respirator, 4°C shaking air incubator, sonicator, 25°C shaking air incubator, Geiger counter, Kimwipes, all equipment for autoradiography.

2.2. Exposure of SW Blots to DNase I Treatment 2.2.1. Solutions 1. Eppendorf tube siliconization solution: 2% (v/v) dimethyldichlorosilane in 1,1,1trichloroethane (BDH). Eppendorf tubes should be silanized by briefly immersing the opened tubes in a beaker containing this solution, pouring off excess solution, and allowing the tubes to dry in air at room temperature. Dimethyldichlorosilane is particularly toxic. Gloves, safety glasses, respirator, and other protective clothing should be worn when handling it and should only be used in a chemical fume hood. 2. Solutions 1, 12, and 15–17 of Subheading 2.1.1. 3. DNase I (stock solution): Dissolve DNase I in 50% glycerol (in distilled, deionized water) to a concentration of 2.5 mg/mL. Store frozen in 10-µL aliquots at –20°C or –70°C. This stock is stable indefinitely. 4. 1 M CaCl2. 5. DNase I reaction buffer: 10 mM MgCl2 and 5 mM CaCl2. Store at room temperature. 6. DNase STOP solution A: 20 mM HEPES·KOH, pH 7.5, 20 mM EDTA, and 0.5% (w/v) SDS. Store at 4°C. 7. 5 M NaCl. 8. DNase-STOP solution B: 60 mM HEPES.KOH, pH 7.5, 0.6 M NaCl, 60 mM EDTA, 1.5% (w/v) SDS. Store at 4°C.

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9. Proteinase K (stock solution): Dissolve Proteinase K in TE (10 mM Tris-HCl, pH 7.4, and 1 mM EDTA) to a concentration of 2.5 mg/mL. Store in aliquots at –20°C. 10. Probe elution buffer: 20 mM HEPES·KOH, pH 7.5, 0.3 M NaCl, 3 mM EDTA, 0.2% (w/v) SDS, and 50 µg/mL Proteinase K. 11. Glycogen (stock solution): Prepare a stock solution of 10 mg/mL glycogen (Sigma G 0885) in distilled, deionized water and store in aliquots at –20°C; glycogen is used as a carrier to promote the precipitation of nucleic acids. 12. Ice-cold 80% (v/v) ethanol. 13. Formamide loading buffer: 90% (v/v) deionized formamide, 1X TBE (see below), 0.025% (w/v) xylene cyanol FF (Sigma), and 0.025% (w/v) bromophenol blue. Store at –20°C after filtering. Formamide is a teratogen; take all safety precautions to avoid contact during manipulations involving this reagent. 14. TBE buffer (5X stock): 445 mM Tris base, 445 mM borate, 12.5 mM EDTA. Dissolve (under stirring for at least 1 h) 272.5 g ultrapure Tris base, 139.1 g boric acid (Sigma), and 23.3 g EDTA·(Na2) dihydrate (Sigma) in 4.5 L of distilled, deionized water. Make up to a final volume of 5 L. It is not necessary to adjust the pH of the resulting solution, which should be around 8.3. Store at room temperature in a large vessel. This stock solution is stable for many months, but it is susceptible to the formation of a precipitate and should be inspected visually from time to time. 15. Fixing solution: 10% (v/v) acetic acid and 10% (v/v) methanol.

2.2.2. Reagents/Special Equipment 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

DNase I (DPFF grade; Worthington, Freehold, NJ). Proteinase K (Boehringer). Absolute ethanol (at –20°C). Single-edged disposable razor blades. Forceps. Siliconized 0.5-mL Eppendorf microcentrifuge tubes. Siliconized 1.5-mL Eppendorf microcentrifuge tubes. Drawn-out Pasteur pipets. Whatman (Clifton, NJ) 3MM paper. Items 16 and 17 in Subheading 2.1.2. Intensifying screen (e.g., Cronex Lightning Plus; DuPont, Wilmington, DE). Additional equipment: beaker, sharp-tip pencil, wet ice, scintillation vials/ counter, vortexer, electronic timer, microcentrifuge (Eppendorf or equivalent), vortexing shaker set at 37°C, Geiger counter, SpeedVac concentrator (Savant, Hicksville, NY), thermostatted heating block at 95°C, plastic tank at gel dimensions (for gel fixing), vacuum gel dryer, and all equipment for autoradiography.

3. Methods 3.1. SW Blotting The SW protocol involves four steps: electrophoretic separation of proteins by SDS-PAGE, electroblotting of the gel-fractionated proteins onto nitrocellu-


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lose (NC) membrane, probing of the blocked blot with the desired DNA probe, and detection of bound DNA by autoradiography of the washed, wet membrane. 1. Prepare and load the sample(s) and protein MW markers onto a standard SDS– polyacrylamide gel (11,12) and electrophorese at an appropriate voltage until the bromophenol blue dye reaches the bottom of the gel. An 8–10% polyacrylamide separating gel is capable to resolve throughout most of the known DNA-binding transcription factor size range. Because the strength of the final signal obtained by the SW technique is proportional to the quantity of protein electrophoresed on the gel, best results are obtained by running the maximum amount of extract that does not overload the gel. For typical 0.5- to 1-mm-thick protein mini-gels, this is usually 30–150 µg of whole-cell-free, crude nuclear, or partially purified extract protein per lane. (See Note 1.) 2. Remove the electrophoresed gel from the glass plates. Assemble a Western blot sandwich, place it in the electrophoresis tank containing an appropriate volume of Western transfer buffer, and electroblot the proteins in the electrophoresed gel onto a NC membrane according to standard Western blotting protocols (11,13). Bear in mind that the best protein transfer is usually achieved by longer transfer times (i.e., 30 V [40 mA] overnight at 4°C). (See Note 2). 3. When transfer is complete, gently peel the NC membrane off the gel, place it in a plastic tray, and gently wash for 10 min with 20–30 mL of SW blocking/renaturation buffer, omitting dried milk (or BSA). 4. Decant the solution and replace it with a sufficiently large volume of SW blocking/renaturation buffer to completely immerse the membrane (usually 30–50 mL). 5. Incubate overnight at 4°C with gentle rocking or shaking to block nonspecific binding sites on the membrane and to allow renaturation of the filter-immobilized proteins. (See Note 3.) 6. Using forceps, transfer the membrane to a fresh plastic tray and gently wash for 5 min with 20–30 mL of SW binding/washing buffer. The membrane can be stored in this buffer at 4°C for up to 1 d before incubation with the DNA probe. 7. Immerse the membrane (using forceps) in a fresh plastic tray containing a radioactive probe mixture consisting of the following: • SW binding/washing buffer (see Note 4). • 5–10 µg (specific activity approx 107 cpm/µg) of an asymmetrically [32P]labeled DNA fragment bearing the recognition site(s) for the sequence-specific DNA-binding factor(s) of interest (see Note 5). • 20 µg/mL nonspecific competitor DNA (see Note 6). To make the probe concentration as high as possible, the volume of SW binding/washing buffer should be the minimum needed to cover the membrane fully (smallest volumes are achieved if the membrane is sealed in a plastic bag). Work behind Perspex or glass shields! 8. Incubate for 2–4 h at room temperature with gentle rocking or shaking. Longer incubation times at a lower temperature may result in a better signal from a poorly binding factor.

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9. Carefully remove the radioactive probe mixture and dispose of it safely (work behind Perspex or glass shields!). 10. Using forceps, transfer the membrane to a plastic tray and wash for 10 min with 100 mL of SW binding/washing buffer on a platform shaker (room temperature). 11. Repeat step 10 two to three times or until the radioactive level of the membrane no longer falls appreciably between washes (this can be monitored by a Geiger counter). (See Note 7.) 12. Place the wet NC sheet between two layers of tightly drawn cling film. With a pad of Kimwipes, push out any trapped air bubbles under the cling film. 13. Expose to X-ray film at 4°C to locate regions of radioactive signal (protein[s]bound probe). Exposure times of 1–3 h are usually sufficient to detect the radioactive species on the membrane. (See Note 8.)

3.2. Exposure of SW Blots to DNase I Treatment The in situ footprinting reaction is done in four stages: localization and excision of the areas on the NC sheet corresponding to protein-bound probe, partial digestion of the individual strip-associated and control (free in solution) DNAs with DNase I, extraction of the protein-bound DNA from the stripimmobilized protein–DNA complex, and analysis of the free and complexed DNA digestion products on a DNA sequencing gel. 1. Following autoradiography, align the NC sheet with the autoradiogram (see Note 8), mark the precise position of radioactive signal(s) with a sharp-tip pencil (this will permit determination of the relative molecular weight of the detected DNA-binding factor[s]), and cut the strip(s) corresponding to radioactive signal(s) with a clean, sharp razor blade. 2. Using forceps, uncover the strip from the cling film and immediately transfer it into a siliconized 0.5-mL Eppendorf tube containing 200 µL of SW binding/washing buffer. 3. Allow the strip to equilibrate for 15 min on ice. Meanwhile, thaw an aliquot of the 2.5-mg/mL DNase I stock solution. 4. Bring the tube to room temperature, place it in a scintillation vial, and determine cpm of the probe retained on the strip by Cerenkov counting. 5. Transfer an equal amount of radioactivity of free probe to a separate, siliconized 0.5-mL Eppendorf tube containing 200 µL of SW binding/washing buffer, and subject it to the same manipulation as its membrane-associated counterpart (step 3). 6. Prepare an appropriate dilution of DNase I in ice-cold distilled, deionized water. Mix thoroughly by inversion and gentle vortexing. 7. Add to both samples 200 µL of DNase I reaction buffer and mix by flicking. Let the tubes stand for 1 min at room temperature. It is not necessary to close the caps on the tubes until addition of the DNase stop solutions (step 10). 8. Add dilute DNase I to a final concentration of 25 ng/mL and quickly distribute by flicking. It is helpful—especially when footprinting several strips—to have all necessary items (pipetmen, buffers, timer, DNase I) in close proximity. The


9.

10.

11.

12. 13. 14.

15.

16.

17.

18.

19.

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smoother this procedure goes, the better (and more reproducible!) the footprint(s) will be. Incubate for 1 min at room temperature (see Note 9). As with solution footprinting protocols, the exposure time for the free-DNA control reaction can be titrated to achieve the cutting intensity profile of the membrane-bound form. Nevertheless, we have found (employing probes of various lengths) that the above combination of digestion time and DNase I concentration generates sufficient cleavage for a good signal-to-noise ratio. Following treatment, remove the strip (with forceps) from the tube and rapidly immerse it in 500 µL of ice-cold DNase-STOP solution A. Leave on ice for 2 min (do not vortex!). Terminate the control reaction (free probe) by adding 200 µL of ice-cold DNase-STOP solution B; vortex thoroughly, spin briefly, transfer into a siliconized 1.5-mL Eppendorf tube, and proceed directly to step 15. Place the strip (using forceps) in a siliconized 0.5-mL Eppendorf tube containing 200 µL of probe elution buffer. Spin briefly in a microcentrifuge to submerge the entire strip. Incubate the tube on a vortexing shaker at 37°C for 2 h. Add 100 µL of distilled, deionized water and vortex the tube vigorously (2 min). Microcentrifuge for 2 min to pellet the NC strip, and transfer the supernatant into a siliconized 1.5-mL Eppendorf tube. A Geiger counter can be used to monitor efficient recovery of radioactivity; typically, >90% of the membrane-bound probe is released by this process. Extract once with phenol/chloroform/isoamyl alcohol (25:24:1; v/v) and once with chloroform/isoamyl alcohol (24:1; v/v). In both cases, mix by vortexing (for 10 s) and spin for 5 min. Transfer the aqueous (top) layer to a fresh siliconized 1.5-mL Eppendorf tube. Precipitate the DNA with cold (–20°C) ethanol in the presence of 10 mM MgCl 2 (added from the 0.5 M stock solution; MgCl2 aids in the recovery of small DNA fragments) and 10 µg carrier glycogen; mix by inversion, spin at 12,000g for 15 min. Remove and discard the supernatant with a drawn-out Pasteur pipet, being careful not to aspirate the DNA (the bottom of the tube can be held to a Geiger counter to check that the DNA pellet remains). Add 800 µL of ice-cold 80% ethanol and rinse the pellet by gently rolling the microcentrifuge tube. Respin for 3 min. Remove and discard the supernatant using a drawn-out Pasteur pipet, taking extreme care not to disturb the tiny, whitish pellet or the area of the tube where the pellet should be located (the DNA pellet frequently adheres only loosely to the walls of the tube). Dry the pellet in a SpeedVac rotary concentrator. Do not allow the drying procedure to continue past the point of dryness because the sample may be difficult to resuspend. Dissolve the pellet in 6–8 µL of formamide loading buffer; pipet the loading buffer onto the upper, inside surface of tube and tap the tube to drop the droplet onto the DNA pellet. Vortex briefly at high speed for approx 15 s and microcentrifuge for 30 s to collect all of the solution to the bottom of the tube.

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20. Transfer to fresh siliconized 1.5-mL Eppendorf tubes and determine the total radioactivity recovered by Cerenkov counting each sample for 1 min in a scintillation counter. 21. Heat samples to 95°C for 3 min to denature DNA and immediately chill in wet ice. 22. Spin briefly to bring the liquid to the bottom of the tubes. Samples can be electrophoresed immediately or stored at –70°C for no more than 24 h after footprinting. 23. Load 1500–2000 cpm of each DNA digestion product (adjust volumes accordingly if necessary; it is essential that a consistent volume of sample be loaded on each lane) onto a pre-electrophoresed, 5–10% denaturing urea (sequencing) gel (see Notes 9b and 10a). Electrophorese in 1X TBE buffer at 60–70 W constant power (for a 34 × 40 cm, 0.4-mm-thick gel) until the marker dye fronts have migrated the appropriate distance in order to visualize the DNA region of interest (11). To determine the location of the transcription factor-binding site(s), the DNase I digests are electrophoresed alongside a Maxam–Gilbert G + A sequencing ladder prepared from the end-labeled footprint probe (14). See Chapter 7 for a fast protocol for preparing such a ladder. 24. Disassemble gel apparatus, carefully lift off one glass plate and soak the gel (still on the second glass plate) in fixing solution for 15 min (see Note 10b). 25. Drain briefly, overlay the gel with two sheets of 3MM paper, and carefully peel it off the glass plate. Cover the gel surface with plastic wrap and dry under vacuum at 80°C for approx 1 h. 26. Expose the dry gel to X-ray film overnight at –70°C with an intensifying screen (a piece of paper placed between the gel and the film will prevent spurious exposure of the film resulting from static electricity). Several different exposures may be required to obtain suitable band densities. 27. Compare lanes corresponding to free and protein-bound DNAs to identify the region(s) of protection; the region(s) of the DNA fragment that is bound by the factor appears as a blank stretch (footprint) in the otherwise continuous background of digestion products.

4. Notes 1. The diversity of properties characteristic of DNA-binding transcription factors imposes an empirical determination of the conditions under which the sample(s) is prepared for SDS-PAGE. In some cases, the reducing agent (2-mercaptoethanol) should be omitted from the sample buffer and, in others, the SDS concentration should be lowered to 0.5%. Furthermore, some DNA-binding proteins may not withstand the sample boiling before loading on the gel. 2. The presence of methanol in the Western transfer buffer may cause a problem during the electrophoretic transfer of some bulky DNA-binding proteins (gel shrinkage reduces pore size). 3. a. The commercially available nonfat dried milk preparations from various suppliers contain large amounts of protein kinase/phosphatase activities; these activities can potentially interfere with binding of the DNA probe to transcription factors whose DNA-binding capacity is known, or suspected, to be subject to


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regulation by inducible phosphorylation/dephosphorylation events. If this is the case, substitute nonfat dried milk in the SW blocking/renaturation buffer for the recommended grade of lipid-free BSA; this particular grade contains only trace amounts of the aforementioned activities and should be preferred as blocking agent (15,16). Moreover, the use of lipid-free BSA results in an even background throughout and enhances the specific signal-to-noise ratio in the DNA-probing step (17). b. By manipulating the conditions for renaturation of the membrane-immobilized proteins (e.g., by incorporating a cycle of protein denaturation and renaturation [16]), the method may be extended to the analysis of proteins resolved in twodimensional gels (18). This offers a powerful and convenient means for studying cell cycle/type/stimulus-dependent DNA–transcription factor interactions and their regulatory roles in gene activity. 4. a. Inclusion of poly(vinyl alcohol) (a molecular crowding agent [volume excluder]) in the buffer decreases the amount of small ions/water available for hydration of any probe dissociated from the immobilized protein matrix and renders the aqueous environment unfavorable for the unbound DNA. Consequently, the effective concentration of the probe is increased and interactions with low binding constants are stabilized. b. It may be important in some cases to supplement this buffer with ZnSO4 (Aldrich, Milwaukee, WI; final concentration 10 µM) if the transcription factor(s) under study is known, or suspected, to contain a zinc-finger domain(s). 5. a. The DNA chosen for probing the protein blot can be a cis-acting regulatory (promoter/enhancer) DNA restriction fragment in the size range of 125–250 bp, with the putative transcription-factor-binding sites located no less than 20–25 bp from the labeled end. This is to ensure that the region of DNA to be investigated for the presence of footprints is capable of being accurately resolved on a sequencing gel. b. A prerequisite for the subsequent DNase I footprinting analysis is the use of a DNA probe that has been labeled on only one strand of the DNA duplex. The labeling of only one strand of a promoter/enhancer restriction fragment can be achieved in a number of ways, such as using T 4 polynucleotide kinase and [γ-32P] ATP (5'-end labeling), the large (Klenow) fragment of E. coli DNA polymerase I and [α-32P] dNTPs (3'-end labeling [“filling in”]), or the polymerase chain reaction (PCR) amplification (19). Preparation of radiolabeled DNA employing any of these methodologies requires about 8 h. A combination of 5' and 3' end-labeled DNA probes allows both strands to be analyzed side by side from the same end of the DNA duplex. c. For optimal sensitivity in the SW procedure, the probe should be of as high a specific activity as possible and highly purified. The latter can be assured by using a nucleic acid-specific, ion-exchange column, such as the Elutip™-d (Schleicher & Schuell) or the NACS prepac cartridge (BRL, Gaithersburg, MD) (11). It is recommended not to store the pure, labeled DNA probe longer than 2–4 d, because the radiation creates nicks in the DNA that will appear as additional bands in the sequencing gel.

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6. Synthetic alternate copolymers, such as poly[dI–dC] · poly[dI–dC] or poly[dA– dT] · poly[dA–dT] (Boehringer or Pharmacia), at similar final concentrations may be more suitable competitors for some DNA-binding transcription factors. 7. Longer washing times at room temperature are detrimental resulting in dissociation of the bound probe, but longer washing times with cold (4°C) SW binding/ washing buffer can reduce background without significant signal loss. To reduce possible low-specificity DNA–protein complexes, the final wash can be performed in cold SW binding/washing buffer with higher salt concentration (i.e., 100–200 mM KCl). 8. If prestained nonradioactive protein MW standards have been used, the edges of the plastic wrap should be marked with pieces of tape labeled with radioactive (or fluorescent) ink (let the ink dots dry completely before exposing to X-ray film!). These marks allow the autoradiogram to be aligned with the protein size markers on the NC sheet (Subheading 3.2., step 1), facilitating calculations on the relative molecular weight of the specific DNA-bound protein species (obtained radioactive signal[s]). If [14C]-methylated protein MW markers have been used, their position will be apparent on the X-ray film without the need to use the radioactive (or fluorescent) ink procedure. 9. a. Although longer digestion times do not enhance background cutting (uncomplexed DNA is minimized), they may lead to substantial deviations from the required “single-hit kinetics” (i.e., on average, each DNA molecule is nicked at most once; this corresponds to nicking approx 30–50% of the DNA molecules). b. Intense bands due to uncleaved, full-length probe should be visible at the top of each lane in the DNA sequencing gel. This aids in determining whether singlehit kinetics are operative and whether equal amounts of total radioactivity are loaded in each lane. 10. a. If the DNA probe is relatively long (i.e., >175 bp) and multiple transcription-factor-binding sites are to be resolved, a gradient or wedge sequencing gel can be used. b. Wedge-shaped gels must be soaked in fixing solution, followed by 5% glycerol for 10 min prior to drying.

References 1. Galas, D. J. and Schmitz, A. (1978) DNase footprinting: A simple method for the detection of protein-DNA binding specificities. Nucleic Acids Res. 5, 3157–3170. 2. Tullius, T. D. and Dombroski, B. A. (1986) Hydroxyl radical “footprinting”: highresolution information about DNA–protein contacts and application to λ repressor and Cro protein. Proc. Natl. Acad. Sci. USA 83, 5469–5473. 3. Kuwabara, M. D. and Sigman, D. S. (1987) Footprinting DNA–protein complexes in situ following gel retardation assays using 1,10-phenanthroline–copper ion: Escherichia coli RNA polymerase–lac promoter complexes. Biochemistry 26, 7234–7238. 4. Johnsrud, L. (1978) Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc. Natl. Acad. Sci. USA 75, 5314–5318. 5. Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to compo-


6.

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nents of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3060. Fried, M. and Crothers, D. M. (1981) Equilibria and kinetics of lac repressoroperator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525. Miskimins, W. K., Roberts, M. P., McClelland, A., and Ruddle, F. H. (1985) Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc. Natl. Acad. Sci. USA 82, 6741–6744. Smith, S. E. and Papavassiliou, A. G. (1992) A coupled Southwestern–DNase I footprinting assay. Nucleic Acids Res. 20, 5239–5240. Polycarpou-Schwarz, M. and Papavassiliou, A. G. (1993) Probing of DNA–protein complexes immobilized on protein-blotting membranes by the chemical nuclease 1,10-phenanthroline (OP)–cuprous ion. Methods Mol. Cell. Biol. 4, 22–26. Polycarpou-Schwarz, M. and Papavassiliou, A. G. (1993) Distinguishing specific from nonspecific complexes on Southwestern blots by a rapid DMS protection assay. Nucleic Acids Res. 21, 2531–2532. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Maxam, A. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499–560. Papavassiliou, A. G., Bohmann, K., and Bohmann, D. (1992) Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors. Anal. Biochem. 203, 302–309. Polycarpou-Schwarz, M. and Papavassiliou, A. G. (1995) Protein–DNA interactions revealed by the Southwestern blotting procedure. Methods Mol. Cell. Biol. 5, 152–161. Papavassiliou, A. G. and Bohmann, D. (1992) Optimization of the signal-to-noise ratio in south-western assays by using lipid-free BSA as blocking reagent. Nucleic Acids Res. 20, 4365–4366. Moreland, R. B., Montross, L., and Garcea, R. L. (1991) Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1. J. Virol. 65, 1168–1176. Lakin, N. D. (1993) Determination of DNA sequences that bind transcription factors by DNA footprinting, in Transcription Factors: A Practical Approach (Latchman, D. S., ed.), IRL, Oxford, pp. 27–47. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., et al. (1987) Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49, 729–739.

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11 Diffusible Singlet Oxygen as a Probe of DNA Deformation Malcolm Buckle and Andrew A. Travers 1. Introduction The DNA double helix is highly malleable and, when constrained, either as a small circle or by the action of a protein, can be readily distorted from its energetically favored conformation. Such distortions may be relatively moderate, as exemplified by smooth bending (which maintains base stacking), or more extreme when this stacking can be disrupted. Deformations of this latter type include kinks, where the direction of the double helical axis is changed abruptly at a single-base step, and localized strand separation, which may be a direct consequence of protein-induced unwinding or of high negative superhelicity in free DNA. Both kinks and localized unwinding can arise transiently during the enzymatic manipulation of DNA by recombinases and by protein complexes involved in the establishment of unwound regions during the initiation of transcription or of DNA replication. The detection of localized lesions in the DNA double helix requires that the bases at the site of the lesion be accessible to a chemical reagent only when the DNA is distorted. Further, because protein-induced distortions are not necessarily sequence dependent, it is desirable that any reagent used for detection possesses minimal selectivity with respect to the base. Additionally the reagent itself should ideally be noninvasive; that is, it should not form a stable noncovalent complex with DNA and thereby possess the potential to perturb the local conformation of the double helix. Other highly desirable attributes for reagents used for this purpose is that they should possess short half-lives and can be generated on demand in situ. These latter characteristics permit the study and detection of transient intermediates in the processes leading to the From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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establishment of complexes competent to initiate replication or transcription or to catalyze recombination. Chemical reagents so far described that specifically target bases in DNA that is locally deformed include dimethyl sulfate, diethyl pyrocarbonate, osmium tetroxide (1), and potassium permanganate (2) (see Chapters 6, 9, and 14). However all of these reagents react selectively with specific bases and are also relatively long-lived. Another reagent used extensively for the detection of locally unwound regions of DNA is copper-o-phenanthroline (3) (see Chapter 7). This compound is a minor-groove ligand, which cleaves the sugar–phosphate backbone as a consequence of free-radical attack on a deoxyribose residue close to the site of binding. One reagent that lacks these shortcomings is oxygen in the singlet state, of which there are two forms with energies of 155 and 92 kJ, respectively. The latter state has a much longer lifetime and can oxidize a variety of unsaturated organic substrates. Typically such a reaction may involve a Diels–Alder-like addition to a 1,3-diene. This highly reactive form of oxygen can be generated by the photochemical excitation of appropriate heterocyclic ring systems that can then promote the conversion of dissolved oxygen in the triplet state to a singlet form. In solution, the singlet state generated in this way has a half-life of approximately 4 ms and can react with accessible DNA bases to form an adduct across a double bond. Once formed, such an adduct sensitizes the sugar– phosphate backbone to alkaline hydrolysis by piperidine, thus permitting the identification of the site of modification (4).

1.1. The Reaction of Singlet Oxygen with DNA The use of singlet oxygen as a reagent for analyzing DNA structure has been pioneered by the groups of Hélène and Austin, who have introduced two general methods of targeting the DNA. In the first case, a DNA ligand is used as a sensitizer for singlet-oxygen production in a manner analogous to the use of copper-o-phenanthroline for the generation of free radicals. Such ligands include methylene blue, which intercalates at sites where the DNA is relatively unwound (4) and also a porphyrin ring covalently linked to a defined DNA sequence designed to target a selected region of double helix (5). In these examples photochemical excitation produces singlet oxygen at the site of the bound ligand and reaction is confined to the immediate proximity of the ligand. A second approach is to use singlet oxygen as a freely diffusible reagent. This use is similar in principle to that of a hydroxyl radical produced by the Fenton reaction (6). In such experiments the singlet oxygen is generated by the irradiation of a complex of eosin with Tris (Fig. 1) and is then free to diffuse. The eosin is irreversibly oxidized in the course of this reaction. Because, however, the half-life of the singlet oxygen is very short, the concentration of the eosin–

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Fig. 1. Reaction cascade for the modification of DNA by singlet oxygen.

Tris complex must be sufficiently high to ensure that singlet oxygen can access potentially reactive sites in the DNA before its reversion to the triplet state. As with any chemical reagent reacting with a set of chemically distinct targets, the rate of reaction of singlet oxygen with the different bases varies. Notably, guanine as the free base reacts up to 100-fold more rapidly than the other nucleic acid bases. However, the rate of reaction of diffusible singlet oxygen with duplex DNA appears not to be primarily determined by the nature of the bases at the target site but rather by their accessibility. In normal B-form DNA, the bases are generally tightly stacked so as to preclude the entry, and hence the reaction, of the reagent between the base pairs. In the structures of DNA oligomers, it is unusual for the average planes of adjacent base pairs to be separated by a roll angle of greater than 10°. This tight structure is reflected in the relative lack of reactivity of DNA in solution toward singlet oxygen. By contrast, when bound by protein, the DNA can be locally unwound (7) or can be kinked so that adjacent base-pairs planes can be inclined by up to 43° relative to each other (8). This deformation of the DNA structure by bound protein would, in principle, be expected to increase the accessibility to singlet oxygen as has been observed both for core nucleosome particles associated with DNA of mixed sequence (4) and for the ternary complex of RNA polymerase and catabolite regulatory protein (CRP) with the lac regula-

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tory region (9). In the latter case, which is the only example for which information is so far available, reaction is observed with all four DNA bases, although there are insufficient sites documented to preclude some base selectivity of the reagent. If the local structure of the DNA is the principal determinant of reactivity, the reagent should be able to access the bases through both the major and minor grooves, as is indeed observed (9). However, the precise range of DNA structures available for reaction with singlet oxygen remains to be established, as does the possible influence of bound protein on sensitizing or quenching the reactivity of the bases. A major advantage of a photoactivated reaction is the ability to produce the reactive species under highly controlled conditions. Both porphyrins and methylene blue can be activated by a continuous laser beam. However, eosin absorbs maximally at 523.2 nm, a wavelength that is close to the 532-nm output from a neodymium–YAG laser. This fortuitous proximity is of particular utility, as it permits the production of singlet oxygen either from an effectively continuous output or from a discrete number of pulses, each of approximately 7 ns duration. This method of activation utilizes the high-energy output of the Nd–YAG laser and also allows the kinetics of the protein-induced structural alterations in DNA to be followed with high precision. It should be noted that because the half-life of singlet oxygen in solution is only 4 ms (10), the time available for reaction with the DNA is essentially limited by the time of irradiation. This short half-life also means that it is unnecessary to terminate any reaction by the addition of a quenching reagent. 2. Materials 1. A neodymium–yttrium–aluminium garnet (Nd-YAG) laser (Spectra-Physics DCR-11) set up as illustrated in Fig. 2 is used to generate a beam of polarized coherent light at a wavelength of 1064 nm. A doubling crystal correctly aligned in the beam path produces a mixture of light at 1064 and 532 nm. A dichroic mirror arranged so that the 532-nm beam is deflected down onto a thermostatted Eppendorf tube containing 20 µL of the sample to be irradiated subsequently separates this mixture. Alternatively, if the volume of the irradiated solution is small, the different wavelengths can be separated by an appropriate arrangement of prisms and the 532-nM beam directed into an Eppendorf tube held horizontally in a metal block maintained at the required temperature. It is essential to obtain an adequate separation of these two wavelengths because even a relatively low proportion of the primary emission at 1064 nm could result in a rapid heating of the sample. The DCR-11 functions at a frequency of 10 Hz each pulse of about 7 ns duration delivering 160 mJ of energy. Other NdYAG lasers are obtainable that can deliver up to twice this energy per pulse. 2. Eosin isothiocyanate is obtained from Molecular Probes. 3. 10 mM Tris-HCl, pH 7.9.

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Fig. 2. Use of a Nd-YAG laser for irradiation of a 20-µL reaction mixture. 4. DNA fragment containing the protein-binding site. The fragment should be labeled at one end with 32P using, for example, end filling with either the Klenow fragment of DNA polymerase or reverse transcriptase (11). The concentration of the stock solution of fragment is typically in the region of 100 µg/mL. 5. An appropriate buffer that is suitable for the protein–DNA complexes is under investigation. Avoid the use of reducing agents. 6. Bovine serum albumin: stock solution 10 mg/mL. 7. Phenol: equilibrated with an equal volume of 0.1 M Tris-HCl, pH 8.0. 8. Absolute ethanol. 9. Piperidine: 0.1 M piperidine is prepared by dilution of redistilled piperidine (10.1 M). 10. 10X TBE: 108 g Tris base, 55 g boric acid, and 40 mL of 0.5 M EDTA (pH 8.0) in 1:1 solution. 11. Polyacrylamide gel: For a 200 bp DNA fragment, a 40 × 20 × 0.04-cm 8% denaturing polyacrylamide gel is used. The gel is prepared by mixing 10 mL of 40%

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Buckle and Travers acrylamide solution (380 g DNA-sequencing grade acrylamide, 20 g N,N'methylenebisacrylamide in 1:1 solution), 5 mL 10X TBE, 23 g urea, deionized water to 50 mL. When the urea is fully dissolved, add 100 µL of 10% ammonium persulfate and mix with rapid stirring, Then, add 60 µL TEMED (N,N,N'N'tetramethylethylene diamine) and mix rapidly. Pour the gel solution between the sealed gel plates and insert a comb with 0.25-cm teeth into the gel solution. Allow to set, remove comb, and clean slots with gel buffer using a Hamilton syringe. Gel running buffer, 1X TBE: 90 mM Tris, 90 mM borate, pH 8.3, and 10 mM ethylenediaminetetracetate (EDTA) pH 8.0 made by dilution of 10X TBE. Gel loading buffer: 95% formamide, 10 mM ethylenediaminetetracetate (pH 8.0), 1 mg/mL xylene cyanol FF, and 1 mg/mL bromophenol blue. X-ray film. 10 mM dithiothreitol (DTT).

3. Methods 3.1. Preparation of Eosin–Tris Complex 1. A 10 mM stock solution of this complex is formed by incubating 10 mM eosin isothiocyanate in 10 mM Tris-HCl, pH 7.9 for 2 h at 37°C, taking care to avoid exposure to light. The eosin isocyanate is diluted from a freshly prepared 100 mM stock solution. 2. The concentration of eosin is estimated from the absorption of the solution at 525 nm (ε523.2 = 25.6 for a 1 M solution) and adjusted by the addition of the appropriate volume of double-distilled water.

3.2. Formation of Nucleoprotein Complexes 1. The first step in the detection of protein-induced deformation of DNA is the formation of a nucleoprotein complex. For example, RNA polymerase (100 nM) and end-labeled fragments of DNA containing the lac UV5 promoter (4 nM) are mixed in a buffer containing 100 mM KCl, 10 mM MgCl 2, 20 mM HEPES (pH 8.4), 3% glycerol, and 100 µg/mL bovine serum albumin in a total volume of 20 µL (9). 2. This mixture is then incubated for 30 min at 37°C.

3.3. Irradiation of the Nucleoprotein Complex 1. Once the nucleoprotein complex has been formed a fresh stock of the eosin–Tris complex is added at an appropriate concentration to 20 µL of the target solution in a small Eppendorf tube. Typically, a final concentration of 50 mM is used (see Note 1). 2. Immediately after the addition of the eosin–Tris complex the whole mixture is irradiated for 20 s at 10 Hz. This corresponds to a total energy dose of 115 J/cm2 (see Note 2). Successful activation of the eosin–Tris complex is apparent by a detectable change in the color of the solution consequent upon a shift in the absorption maximum from 523 to 514 nm on oxidation.

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3. Although the half-life of singlet oxygen is sufficiently short to obviate the need to remove excess reagent, it is advisable to add a quenching agent such as dithiothreitol immediately on cessation of irradiation to minimize any secondary radical reactions. Typically, 1 µL of 10 mM dithiothreitol is added to the irradiated solution.

3.4. Detection of Sites of Reaction with Singlet Oxygen 1. After irradiation and quenching, 30 µL double-distilled water is added to each sample. 2. Fifty microliters of a phenol freshly prepared by equilibration of melted phenol with an equal volume of 0.1 M Tris-HCl is then added. 3. After mixing with a vortex mixer, the samples are centrifuged for 1 min in a bench-top microcentrifuge at 5000g to separate the aqueous and organic layers. 4. The upper aqueous layer is removed with an automatic pipet to a clean Eppendorf tube. 5. Three to four volumes of ethanol at 0°C are then added and the samples placed in a dry ice/ethanol bath for 1 h. 6. Centrifuge the samples for 15 min at 5000g in a bench-top centrifuge. 7. Remove the ethanol using an automatic pipet. 8. Dry the samples in a centrifugal evaporator. 9. Resuspend in 100 µL of freshly prepared piperidine solution. 10. Heat at 90°C for 30 min (see Note 6). 11. Sites of cleavage are determined by separation on polyacrylamide gels (typically 40 cm, run at 60 W constant power until the xylene cyanol FF marker has migrated 23 cm into gel) followed by autoradiography (typically 2–24 h exposure depending on the specific activity of the labeled DNA fragment). A typical result is shown in Fig. 3. Cleavage at a particular site results in the generation of a band of defined length. Standard Maxam and Gilbert sequencing reactions (12) can be performed and loaded on the same gel to identify the cleavage sites.

4. Notes 1. Because the half-life of singlet oxygen is short, the average path length for diffusion is also short. Consequently, to ensure an adequate rate of reaction, the eosin– Tris concentration must be sufficiently high to ensure that all potential targets in the DNA are accessible to the reactive entity. 2. Although the energy dose used during irradiation may appear to be substantial, it should be borne in mind that even with a high-intensity laser, each pulse delivers only 160 mJ and consequently, full saturation of the system requires a considerable pulse repetition rate. 3. For optimum reactivity, it is essential that radical scavengers such as mercapto– groups should, as far as possible, be rigorously excluded from the reaction mixture, as they would effectively prevent any singlet oxygen from arriving at its target site. For the same reasons, the concentrations of alcohols such as glycerol should be kept as low as possible. 4. Ideally, protein–DNA complexes should be insensitive to the presence of the nonirradiated eosin–Tris complex. However, it has been observed that the half-

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Fig. 3. Reaction of singlet oxygen with a binary complex of E. coli RNA polymerase with the lac UV5 promoter. The figure shows an autoradiograph of the pattern of reactivity on the transcribed strand in the presence and absence of RNA polymerase. Note that the bands visible in the DNA only lane result from piperidine cleavage at (Py)3 sequences and their occurrence is independent of both irradiation and the presence of eosin–Tris.

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life of certain complexes, in particular the binary CAP–DNA and RNA polymerase–DNA complexes, is reduced by approximately an order of magnitude with the sensitizer present (9). For stable complexes in which the protein has a long residence time this effect does not significantly interfere with the detection of DNA deformations because the time of irradiation is short relative to the stability of the complex. At this time, it is unclear whether this effect is general or is restricted to particular complexes. Nevertheless, it is essential to determine the stability of complexes under study under the precise conditions corresponding to those prevailing during irradiation. The method of choice is gel retardation (electrophoretic mobility shift assay). 5. The short time of irradiation allows the use of the singlet-oxygen reaction in kinetic studies. Here again, to prevent any perturbation of an enzymatic manipulation of DNA, it would be necessary to add the eosin–Tris complex immediately prior to irradiation after the reaction under study had proceeded for the required time. For this purpose, a rapid-mixing device would be necessary. 6. To obtain sharp bands on polyacrylamide gels, it is advisable for the samples to be transferred to clean Eppendorf tubes immediately prior to the evaporation of piperidine. Removal of piperidine in a centrifugal evaporator should also be carried out as rapidly as possible, and any form of heating should be avoided, as this increases the nonspecific background cleavage of DNA by piperidine.

References 1. Lilley, D. M. J. and Palecek, C. (1984) The supercoil-stabilised cruciform of colE1 is hyper-reactive to osmium tetroxide. EMBO J. 3, 1187–1195. 2. Sasse-Dwight, S. and Gralla, J. D. (1989) KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J. Biol. Chem. 264, 8074–8081. 3. Sigman, D. S., Spassky, A., Rimsky, S., and Buc, H. (1985) Conformational analysis of lac promoters using the nuclease activity of 1,10-phenanthroline–copper ion. Biopolymers 24, 183–197. 4. Hogan, M. E., Rooney, T. F., and Austin, R. H. (1987) Evidence for kinks in DNA folding in the nucleosome. Nature 328, 554–557. 5. Le Doan, T., Perrouault, L., Hélène, C., Chassignol, M., and Thuong, N. T. (1986) Targeted cleavage of polynucleotides by complementary oligonucleotides covalently linked to iron-porphyrins. Biochemistry 25, 6736–6739. 6. Tullius, T. D., Dombroski, B. A., Churchill, M. E. A., and Kam, L. (1987) Hydroxylradical footprinting: a high resolution method for mapping protein–DNA contacts. Methods Enzymol. 155, 537–558. 7. Ansari, A. Z., Chael, M. L., and O’Halloran, T. V. (1992) Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87–89. 8. Schultz, S. C., Shields, S. C., and Steitz, T. A. (1991) Crystal structure of a CAP– DNA complex: the DNA is bent by 90°. Science 253, 1001–1007. 9. Buckle, M., Buc, H., and Travers, A. A. (1992) DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen. EMBO J. 11, 2619–2625.

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10. Rougée, M. and Bensasson, R. V. (1986) Détermination des constantes de vitesse de désactivation de l’oxygène singulet (1D7) en presénce de biomolécules. Comp. Rend. Acad. Sci. Paris 302, 1223–1226. 11. Travers, A. A., Lamond, A. I., Mace, H. A. F., and Berman, M. L. (1983) RNA polymerase interactions with the upstream region of the E. coli tyrT promoters. Cell 35, 265–273. 12. Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Methods Enzymol. 155, 560–568.

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12 Ultraviolet-Laser Footprinting Johannes Geiselmann and Frederic Boccard 1. Introduction 1.1. Measurement of DNA–Protein Interactions In Vitro A large number of processes within the cell, in particular the regulation of gene expression, rely on the binding of proteins to specific sites on the DNA. A primary ingredient to understanding these processes is the characterization of the protein–DNA interaction (1). Such a characterization consists in determining the position of the binding site on the DNA and measuring the affinity of the protein for this recognition site. A wide variety of footprinting techniques can accomplish this task (2,3).

1.2. Footprinting Techniques These techniques involve the reaction of a footprinting reagent (in the largest sense) with DNA and the subsequent localization and quantification of the resultant DNA modification. Commonly used footprinting reagents include DNase I, KMnO4, or dimethylsulfate (DMS) (2,3); see Chapters 3–14. Most of these methods require extended incubation times of the reagent with the DNA– protein complex, which may lead to artifacts if the footprinting reagent modifies the complex. For example, DMS may react with the protein; DNase I relaxes a supercoiled plasmid, which may destabilize the DNA–protein complex under investigation. UV-laser footprinting eliminates several of these disadvantages, but also creates others (see Subheading 1.4.).

1.3. Principle of UV-Laser Footprinting The principle of ultraviolet (UV)-laser footprinting is schematized in Fig. 1. The sample containing the nucleoprotein complex is irradiated with a short (less than 10 ns) pulse of UV-laser light. An identical sample, but lacking the From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Fig. 1. The principle of UV-laser footprinting. The double-stranded DNA is represented by the double line, and the protein by the ellipse. (1) A sample of DNA alone, or the DNA–protein complex is irradiated with one pulse of UV-laser light. The bases can undergo intramolecular photoreactions, react with the solvent (symbolized by the circle), or form a crosslink with the bound protein (symbolized by the line connecting DNA and protein). Only the reactions of the top strand are shown in the figure. (2) The samples are denatured, a radioactively labeled primer (line with diamond) is annealed to one stand of the DNA (the top strand) and extended using a DNA polymerase (dotted line). The primer extension stops at damaged bases or at the end of the fragment. (3) The primer extension products are analyzed on a sequencing gel, along with a dideoxy-sequencing reaction using the same primer. The location of the photoreactions are marked with an arrow, the crosslink and the runoff are indicated.

protein, is treated in parallel in the same way. The conditions are adjusted such that the number of photons delivered onto the sample exceeds the number of absorbing molecules. The nucleic acid bases are excited and undergo photoreactions, the nature of which depend exquisitely on the local environment of the bases. The possible reactions include intrastrand reactions of consecutive bases (the formation of thymine dimers is the most prominent such reaction), interstrand reactions (although their quantum efficiency is too low to contrib-


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ute to footprinting signals), reactions with solvent molecules (e.g., with H2O), and crosslinks with the protein (4–8). In general, it is not possible, but neither is it necessary, to determine the nature of the photoreaction at a particular base. Because the photoreactions are highly sensitive to the local environment of the DNA, binding of a protein changes this environment and thus produces a footprint (i.e., a difference between the DNA photoreactivity in the absence and presence of the protein). In the example of Fig. 1, the photoreaction at the left extremity of the DNA fragment remains constant because the protein does not change the local environment of this base. However, the presence of the protein prevents a photoreaction toward the right end of the fragment (perhaps by excluding water from the vicinity of the base) and favors a new photoreaction with an amino acid side chain, resulting in a covalent bond, a crosslink, between the protein and the DNA. All such reactions modify the nucleotide base and, hence, impede the progression of DNA polymerase during subsequent replication of the damaged DNA strand. Arrest of the polymerase is detected in a primer extension reaction by the appearance of shortened replication products, which then serve to localize the modified base.

1.4. Advantages and Disadvantages of UV-Laser Footprinting Ultraviolet-laser footprinting circumvents many potential artifacts of more classical techniques by trapping the complex under investigation at the time of irradiation. The signal is acquired very rapidly (on the order of microseconds) (i.e., faster than typical rearrangements of a DNA–protein complex [on the order of milliseconds]), and the footprint thereby freezes the initial state of the complex (9). Laser light, as opposed to ordinary UV light, is needed in order to limit the “incubation time” to several ns while providing a sufficient number of photons to excite all nucleotide bases of the sample. The rapidity of signal acquisition allows one to obtain kinetic structural signals by irradiating a complex at different times after mixing the components. The technique has the further advantage that it can be transposed to in vivo experiments in a straightforward manner because UV light readily penetrates bacterial cells. However, because of the limited sample size of in vivo experiments, the present version of the technique is only suitable for studying DNA–protein interactions in bacteria when using a binding site carried on a multicopy plasmid. Using the UV-laser technology, it is possible to follow the kinetics of the assembly of DNA–protein complexes. This technique can be used to determine the order in which protein–DNA contacts are established in a multiprotein–DNA complex. Proteins and DNA are mixed at time zero and the sample is irradiated at a specific time interval after mixing (10). Modern mixing techniques allow a time resolution on the order of a several milliseconds (11).

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The main disadvantage of UV-laser footprinting is the unpredictability of the footprinting signal. It should be noted that the footprint is not the result of the protein shielding the DNA from the UV irradiation. The presence of the protein merely changes the probability of certain photoreactions by excluding solvent, changing the conformation of the DNA, or juxtaposing a reactive amino acid and a particular base. A structural interpretation of the footprinting signal is therefore, in general, not possible. A more practical limitation of the technique is the need for a relatively expensive laser. We describe the experiment for the particular case of the binding of an Escherichia coli protein, the integration host factor (IHF), to one of its specific binding sites, the yjbE site (12), in vitro and in vivo. IHF is a small, heterodimeric protein (molecular weight [MW] of the dimer is approx 19 kDa) that binds to specific sites on the DNA (for a review, see ref. 13). Upon binding to DNA IHF bends the DNA by about 180º (14). This DNA bending gives rise to a very strong UV-laser footprinting signal, probably because two consecutive pyrimidines are brought into optimal alignment for the formation of a pyrimidine dimer (15). Variations of this basic protocol, applicable to the study of any DNA–protein interaction, are described in Subheading 4. 2. Materials 1. Ultraviolet-laser: The most commonly used lasers are YAG lasers, e.g., the Spectra-Physics Quanta-Ray GCR series lasers, which emit infrared light of 1064 nm. Two consecutive passes through a frequency-doubling crystal yields high-intensity light of 266 nm, which is sufficiently close to the absorption maximum of nucleic acid bases. Frequency doublers are a standard add-on for virtually all commercially available YAG lasers. The laser power should be between 30 and 50 mJ per pulse at 266 nm and a pulse duration of 5 ns is standard. The energy of one 30-mJ pulse represents about 4 × 1016 photons (i.e., 67 nmol photons). 2. Power meter to measure the energy of the laser beam. 3. Thermostated water bath. 4. Spectrophotometer. 5. Water pump and 0.45-µM filter device for washing E. coli cells. 6. Phosphoimager: A phospho-storage device is ideal for quantifying sequencing gels. The most commonly used instruments are sold by Molecular Dynamics, Fuji, or by Bio-Rad. 7. IHF binding buffer: 50 mM Tris-HCl, pH 7.5, 70 mM KCl, 7 mM MgCl2, 3 mM CaCl2, 1 mM EDTA, 10% glycerol, 200 mg/mL bovine serum albumin (BSA), and 1 mM β-mercaptoethanol. 8. IHF: working stock solution at 3–5 µM in 50 mM Tris-HCl, pH 7.4, 800 mM KCl, 40 mM K-phosphate, 2 mg/mL BSA, and 10% glycerol. 9. Plasmid pBluescriptII (Strategene) containing the IHF binding site cloned into the multicloning site (MCS). Stock solution in water at 100 nM.


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10. Primer extension reaction. Annealing buffer: 1 M Tris-HCl, pH 7.6, 100 mM MgCl2, and 160 mM dithiothreitol (DTT). Elongation mix for 2 µL: 2 U of T7 DNA polymerase in 2.4 mM of each deoxyribonucleotide, 3 mM Tris-HCl, pH 7.5, 0.75 mM DTT, 15 mg/mL BSA, and 0.75% glycerol (see Subheading 3. for the annealing and elongation steps). Stop solution: 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol. 11. Sequencing reaction. Enzyme dilution buffer: 20 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mg/mL BSA, and 5% glycerol. Stop solution: 95% formamide, 1 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol. 12. Sequencing gel: Sequencing reactions are analyzed on 40-cm-long denaturing (7 M urea) 8% polyacrylamide (ratio acrylamide:bis acrylamide 19:1) gels in 90 mM Tris–borate, and 2 mM EDTA (TBE), and TBE used as running buffer. Gels were transferred onto Whatman (3MM Chr) paper and dried at 80°C for 40 min in a gel dryer (Bio-Rad model 583) linked to a vacuum pump. 13. Extraction of plasmid DNA. Solution I: 100 mM Tris-HCl, pH 7.5, 10 mM EDTA, 400 µg/mL RNase I. Solution II: 0.2 N NaOH, and 1% sodium dodecyl sulfate (SDS) made freshly. Solution III: 3 M potassium, and 5 M acetate solution, made by adding 11.5 mL of glacial acetic acid and 28.5 mL of H2O to 60 mL of 5 M potassium acetate. 14. LB and minimal M9 media are used to grow and wash E. coli cells, respectively (16).

3. Methods 3.1. In Vitro UV-Laser Footprinting 1. Arrange the laser beam, using appropriate mirrors, such that it is directed vertically into a water bath. 2. Align, and fix firmly, an Eppendorf holder in the water bath such that the laser beam enters precisely in the center of an open Eppendorf tube. A piece of black paper stuck into the bottom of the Eppendorf tube can help align the tube with the laser beam; the impact of the laser light is very audible and “burns” the site of impact, whitening the otherwise black paper. 3. Operate the laser in repetition mode (see Note 1) for at least 10 min and adjust the doubling crystals to obtain a laser power at 266 nm of at least 30 mJ per pulse. This laser power is measured during the warm-up period with an appropriate power meter, before the actual footprinting reaction. 4. Incubate IHF at the desired concentration with 5 nM plasmid DNA in 40 µL binding buffer for 20 min at 25°C (see Note 2). It is best to use a flat-bottomed Eppendorf tube, but regular 1.5-mL Eppendorf tubes are adequate. The laser beam generally has a diameter of 5 mm, and for maximal use of the light energy, the sample should have roughly the same dimensions. Care should be taken to ensure that all of the sample is irradiated by the laser beam. 5. Place the sample under the laser beam and irradiate with one pulse of UV-laser light. It is best to operate the laser in repetition mode (i.e., continuously emitting around 10 pulses per second). Most lasers also have the possibility to emit a single pulse of light. However, the power of such a pulse is not very well con-

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Geiselmann and Boccard trolled, because the yield of the doubling crystals is extremely sensitive to temperature. Continuous emission of laser pulses at a frequency of about 10 Hz ensures a constant temperature of the crystals and therefore a stable pulse energy. An electronically controlled shutter is used to obstruct the beam. The shutter opening is coordinated with the emission of the laser pulses to ensure that only one pulse of laser light passes for each opening of the shutter. After irradiation, remove the protein from the irradiated DNA by incubating with 50 µg/mL proteinase K for 15 min at 50°C. Extract the samples with half a volume of a phenol/chloroform solution (made by adding equal volumes of phenol pH 8 and chloroform), precipitate with 2 vol of ethanol, and resuspend the DNA in 18.5 µL of H2O. Primer extension (see Note 3): Add 2 µL of a solution of 0.2 µM radiolabeled primer (5'-[32P] labeled using T4-kinase) to 18.5 mL of DNA. (Increasing the primer concentration beyond this twofold excess over template will increase the strength of the primer extension signals only marginally.) Denature the samples by heating to 100°C for 3 min and chill on ice for 5 min. After the addition of 2.5 µL of annealing buffer, incubate the samples for 3 min at 50°C (to anneal primer) and chill again for 5 min on ice. Add 2 µL of the elongation mix (see Note 4) and incubate the reaction for 10 min at 37°C. The mix is prepared freshly but can be kept on ice for several hours. Precipitate the DNA by adding 150 µL of ethanol and incubating for 10 min at –20°C. Centrifuge for 10 min at full speed (about 12,000g) in a microcentrifuge. Resuspend the DNA in 10 µL of loading dye. Analyze 3 µL by gel electrophoresis on a denaturing 8% polyacrylamide sequencing gel. After electrophoresis, transfer the gel onto Whatman paper and vacuum dry at 80°C for 40 min. To determine precisely the location of the footprint, a reference ladder is generated by sequencing the same plasmid DNA using the same radiolabeled primer (see Note 5). Denature 15 nM of plasmid DNA in a volume of 8 µL by heating to 100°C for 2 min. Chill on ice for 5 min. Add 1 µL of radiolabeled primer (0.25 µM) and 1 µL of annealing buffer. Incubate for 3 min at 50°C. Chill annealing reaction on ice for 5 min. Add 2.8 µL of each Deaza G/A T7 Sequencing™ Mixes (Pharmacia) to four termination reaction tubes and prewarm at 37°C. Dilute 1 µL of T7 DNA polymerase with 4 µL of enzyme dilution buffer. Add 2 µL of diluted T7 DNA polymerase to the annealing reaction. Dispense 2.8 µL of annealing reaction in each of the termination tubes. Incubate for 5 min at 37°C. Add 4 µL of the stop solution. Expose the dried gel to a phospoimager screen overnight and scan the screen using the phospho-imager and its associated software (e.g., ImageQuant from Molecular Dynamics). Deduce a line profile of the different lanes using the Phospho-Imager software. Transfer the data to Microsoft Excel and superimpose the scans on the same graph in order to visualize and quantify the footprint (see Notes 6–11).


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3.2. In Vivo UV-Laser Footprinting 1. Grow cultures of IHF+ (W3110) and IHF– (W3110 hip) strains (12) transformed with the plasmid carrying the ihf site in LB medium to the desired OD600 (0.6 or to saturation). 2. Wash the cells in minimal M9 medium (optically transparent buffer), resuspend in minimal M9 medium to a final OD600 of 1, and incubate the cells at 37°C (see Note 12). 3. Irradiate as described above a large number (40–60), of 50-µL cell aliquots (see Note 13). Freeze the cells immediately after irradiation in a dry-ice bath. 4. Pool the cells and extract plasmid DNA from 2–3 mL of cells using the following alkaline lysis procedure. Centrifuge the bacteria for 5 min in a tabletop Eppendorf centrifuge. Resuspend the pellet in 100 µL of solution I and add 100 µL of solution II. Mix by inversion several times. Add 100 µL of solution III and mix by inverting the tube several times. Centrifuge the tubes at full speed in a tabletop Eppendorf centrifuge (>10,000g) for 5 min. Transfer the supernatant to a clean tube and add 210 µL of isopropanol. Incubate for 5 min at room temperature and centrifuge the tubes at full speed for 10 min. Wash the pellet with 70% ethanol and dissolved the DNA in 37 µL of H2O. 5. For primer extension, add 2 µL of a solution of 0.2 µM primer (5'-[32P] labeled) to 18.5 mL of DNA and process and analyze the samples in the same way as for the in vitro reactions, steps 7–13 of Subheading 3.1. (see Notes 10 and 11).

4. Notes 1. Laser setup. As mentioned in the Subheading 3. in order to obtain a stable laser power it is best to operate the laser in repetition mode. If a single-pulse mode is used the energy of a particular pulse is ill-defined and the absence of a footprinting signal may simply be the consequence of diminishing laser power. All lasers provide an electrical signal that allows external equipment to be coordinated with the laser pulse. To our knowledge, shutters are not commercially available. However, it is an easy task for a good mechanics shop to construct such a shutter. We used a shutter made of a small sheet of blackened Teflon obstructing a hole of approx 8-mm in diameter through which the laser beam had to pass in order to reach the sample. Any material can be used, but it should be kept in mind that the laser will eventually burn a hole into the material and that it is best to use a black material in order to minimize hazardous reflections of the laser light. A simple electronic circuit controlled the opening of the shutter by activating an electomagnet that pulled the Teflon sheet (via an attached piece of metal) away from the hole. After the light had passed, the current to the magnet was cut and a spring pulled the sheet back over the hole. 2. The binding buffer described is the standard buffer used for measuring DNA binding of IHF. Other proteins may require different buffer conditions. The buffer may be adjusted with certain limitations. The salt concentration should not be too low in order to avoid nonspecific binding of the protein to DNA. The buffer should not include a high concentration of reagents that absorb at 266 nm. For

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Geiselmann and Boccard example, the interaction of the cyclic AMP receptor protein (CRP) with DNA requires the presence of cAMP in the buffer (17). Keep the concentration of such nucleotides below 100 µM. As mentioned in Subheading 3., one laser pulse contains the equivalent of about 100 nmol in photons. A typical reaction volume is 50 µL; therefore 100 µM ATP absorbs about 5 nmol of photons. A more physiological concentration of ATP in the millimolar range would dramatically decrease the yield of the photoreaction. The procedure describes the footprinting reaction for only one strand of DNA. Evidently, the other strand can be analyzed in the same way using the appropriate primer. The primers should be chosen such that the region of interest is within 200 nucleotides from the primer. Most photoreactions have a quantum efficiency below 1%, the formation of thymine dimers reaching several percent. Therefore, on average, there will be roughly 1 photoreaction per 100 base pairs (bp). Considering only the 100-bp region downstream of a primer assures single-hit conditions. If the DNA carries too many photoreactions, the primer extension will stop at the first defect and the signal of a photoreaction further downstream will pass undetected. The detection of photoreactions. All DNA polymerases are very sensitive to damaged bases. It is not important to use T7 DNA polymerase in the primer extension reaction, any other DNA polymerase (e.g., Klenow, Taq) gives equivalent signals. However, signals obtained with different polymerases may not necessarily be identical because some may be more sensitive to particular photodamaged bases than others. Even RNA polymerases can be used if the template harbors an appropriate promoter. We have successfully used T7 RNA polymerase to transcribe the region of interest from a T7 promoter located on the vector DNA. The major signal of the IHF footprint remained unchanged, but, instead of a single band, T7 RNA polymerase generates a doublet of bands (15). To determine the location of termination sites precisely, we generated a reference DNA ladder consisting of a sequencing reaction of the same DNA region (see Subheading 3.). In general, it is assumed that the primer extension reaction of the irradiated DNA stops just before the modified base. For example, if the sequence of the DNA read on the gel is 5'-GGAC-3' and the primer extension reaction of the irradiated DNA shows a band at the position of the A in the sequencing reaction (run in parallel on the gel), then the photoreaction most likely took place on the following base pair (the C in the above sequence). Because the primer extension reaction reads the opposite strand, the photoreaction actually damaged the G marked with an asterisk: 5'-GGAC-3' 3'-CCTG*-5' There are several possible reasons for not detecting a UV-laser footprinting signal. The most obvious problem is that the protein does not bind to the DNA. Generally, we verify binding by a gel retardation assay. The second reason may be that protein binding does not change the photoreactivity of the bases in the recognition site. This is the case, for example, for CRP, which yields very weak


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signals in UV-laser footprinting despite a strong interaction measured by other techniques (J. Geiselmann, unpublished results). Because photoreactivity depends on the sequence, a particular site might not be photosensitive; for example, the main IHF signal had not been observed for the ssb site, probably because the sequence of this site does not contain the highly reactive TC pyrimidine doublet (15). A trivial reason for not detecting a photoreaction is that the laser did not hit the sample. The best control, and a control to include in all laser footprinting reactions, is to analyze the primer extension reaction of DNA alone. Two DNA-alone samples should always be included: one irradiated sample and an identical sample that has not been exposed to UV-laser light. The irradiated sample should yield readily visible bands all along the lane (Fig. 2, lane f), whereas the nonirradiated sample should show no elongation arrests (Fig. 2, lane e). The irradiated DNA does not give any elongation arrests with T7 DNA polymerase. Verify the primer extension mix. Perform a control primer extension reaction using the nonirradiated plasmid template, but cut about 100 bp downstream of the primer with a convenient restriction enzyme. Primer extension using this template should give a very strong band corresponding to the elongation reaction reaching the end of the fragment. Artifactual footprinting signals. A contaminated template preparation or a damaged DNA template could be misinterpreted as giving a footprinting signal. It is very important to verify that the nonirradiated template does not produce any bands during the primer extension reaction. This is particularly important for in vivo reactions because the plasmid preparation could partially damage the plasmid and lead to artifactual bands (Fig. 2, lanes c and d). Comparing signals obtained under different conditions in vitro, or comparing in vitro to in vivo signals. The efficiency of sample preparation or of the primer extension reaction can vary from sample to sample. In order to compare different lanes we run a small portion (10%) of the primer extension reactions on a sequencing gel and quantify the lanes using a phosphoimager. We then load the same samples on a second sequencing gel, but equilibrating the amounts loaded according to the signal intensities on the first gel. For example, if one of the reactions was only half as efficient as the other ones, we load two times more of this sample on the second gel. This readjustment should not be allowed to exceed a factor of 2. Quantifying lanes. Once an intensity equilibrated gel exposure is obtained, we obtain line profiles of all lanes using the Phospo-Imager software and compare lanes by superimposing the line profiles in Microsoft Excel. Remaining small (several percent) differences in the intensities of the lanes should be normalized by multiplying the scans with a scaling factor between 0.9 and 1.1 that is determined subjectively by the user in such a way that the global patterns superimpose. Lanes containing a high background of nonspecific radioactivity cannot be quantified with confidence. It is important to keep in vivo samples at the physiological temperature (37°C for E. coli) to ensure optimal binding.

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Fig. 2. UV-laser footprinting. (A) Primer extension profile of a UV-laser footprinting experiment showing the binding of IHF to a specific binding site. The four lanes on the left, labeled TCGA, are a sequencing reaction using the same primer as the one used for the primer extension of the UV-laser footprinting reaction (only the T lane is clearly visible on the picture). Increasing amounts of IHF (0–200 nM, lanes f to l) are incubated with 5 nM plasmid and footprinted in vitro, as described in Subheading 3. The arrow points to the major footprinting signal. Lane e is identical to lane f, but the DNA has not been irradiated. The in vivo reactions are carried out as described in the protocol. Lane a is derived from wt cells expressing IHF, lane b is a footprinting reaction from a strain lacking IHF.


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The control lanes c and d show that the preparation of the plasmid DNA is sufficiently clean for an efficient primer extension and that the bands seen in lanes a and b are due to the UV irradiation. (B) Superposition of line profiles from lanes f, h, and l, corresponding to the indicated concentrations of IHF. The intensities of the bands are in arbitrary units. (C) An equivalent superposition of the in vivo profiles and the in vitro profile corresponding to the 50-nM IHF lane shows that E. coli contains roughly the same amount of free IHF in stationary-phase cells as was present in the in vitro sample using 50 nM (total) IHF. As expected, the footprint in a strain lacking functional IHF shows the same profile as DNA alone in the in vitro reaction.

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13. In vivo footprinting. Primer extension of the irradiated template can only be performed in vitro. It is therefore necessary to extract the irradiated DNA from the bacteria. Our current technology allows the measurement of protein binding to specific binding sites carried on a multicopy plasmid. In principle, a primer extension reaction on chromosomal DNA should work as well. In practice the signals obtained from chromosomal DNA are too weak. Increasing the number of samples irradiated does not remedy the problem. Because of the large excess of chromosomal DNA with respect to the primer extension product, we observe abnormal migration of the band in the sequencing gel. The sample for in vivo UV-laser footprinting must be prepared such that a single pulse of the laser (typically about 30 mJ per pulse, corresponding to 4 × 1016 photons [i.e., 67 nmol of photons]) delivers more photons than there are absorbing molecules in the sample. For an in vivo experiment, the absorbing molecules are mostly made up of cellular DNA and RNA, as well as free nucleoside phosphates. An upper estimate of the concentration of absorbing molecules within an Escherichia coli cell is about 100 mM, corresponding to 6 × 108 absorbers per cell. Because a single pulse delivers 4 × 1016 photons and because we want an excess of photons over absorbers, we want to irradiate less than about 108 E. coli cells per pulse. This numbers corresponds to about 100 µL of a suspension at 1 OD600. A large number of 50-µL samples are therefore irradiated and the cells are frozen immediately after irradiation.

References 1. von Hippel, P. H. and Berg, O. G. (1986) On the specificity of DNA–protein interactions. Proc. Natl. Acad. Sci. USA 83(6), 1608–1612. 2. Sauer, R. T. (1991) Protein–DNA interactions, in Methods in Enzymology, vol. 208, Academic Press, San Diego, CA. 3. Jost, J.-P. and Saluz, H. P. (1991) A laboratory guide to in vitro studies of proteinDNA interactions, in Biomethods, vol. 5, Birkhäuser Verlag, Basel. 4. Hockensmith, J. W., Kubasek, W. L., Vorachek, W. R., and von Hippel, P. H. (1993) Laser cross-linking of proteins to nucleic acids. I. Examining physical parameters of protein–nucleic acid complexes. J. Biol. Chem. 268, 15,712–15,720. 5. Pashev, I. G., Dimitrov, S. I., and Angelov, D. (1991) Crosslinking proteins to nucleic acids by ultraviolet laser irradiation. Trends Biochem. Sci. 16, 323–326. 6. Panyutin, I. G., Kovalsky, O. I., and Budowsky, E. I. (1989) Irradiation of the template with high-intensity (pulse-laser) ultraviolet light results in DNA–polymerase termination events at deoxyguanosine residues. FEBS Lett. 258, 274–276. 7. Menshonkova, T. N., Simukova, N. A., Budowsky, E. I., and Rubin, L. B. (1980) The effect of high intensity ultraviolet irradiation on nucleic acids and their components. Cleavage of N-glycosidic bond in thymidine, adenosine and 2'-deoxyadenosine. FEBS Lett. 112, 299–301. 8. Matsunaga, T., Hieda, K., and Nikaido, O. (1991) Wavelength dependent formation of thymine dimers and (6–4) photoproducts in DNA by monochromatic ultraviolet light ranging from 150 to 365 nm. Photochem. Photobiol. 54, 403–410.


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9. Hockensmith, J. W., Kubasek, W. L., Vorachek, W. R., Evertsz, E. M., and von Hippel, P. H. (1991) Laser cross-linking of protein-nucleic acid complexes. Methods Enzymol. 208, 211–236. 10. Eichenberger, P., Dethiollaz, S., Buc, H., and Geiselmann, J. (1997) Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting. Proc. Natl. Acad. Sci. USA 94, 9022–9027. 11. Buckle, M., Pemberton, I. K., Jacquet, M. A., and Buc, H. (1999) The kinetics of sigma subunit directed promoter recognition by E. coli RNA polymerase. J. Mol. Biol. 285, 955–964. 12. Murtin, C., Engelhorn, M., Geiselmann, J., and Boccard, F. (1998) A quantitative UV laser footprinting analysis of the interaction of IHF with specific binding sites: re-evaluation of the effective concentration of IHF in the cell. J. Mol. Biol. 284, 949–961. 13. Nash, H. A. (1996) The HU and IHF proteins: accessory factors for complex protein-DNA assemblies, in Regulation of Gene Expression in Escherichia coli (Lin, E. E. C. and Lynch, A. S., eds.), R.G. Landes Company, Austin, TX, pp. 149–179. 14. Rice, P. A., Yang, S., Mizuuchi, K., and Nash, H. A. (1996) Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell 87, 1295–1306. 15. Engelhorn, M., Boccard, F., Murtin, C., Prentki, P., and Geiselmann, J. (1995) In vivo interaction of the Escherichia coli integration host factor with its specific binding sites. Nucleic Acids Res. 23, 2959–2965. 16. Miller, J. H. (1992) A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 17. Buckle, M., Buc, H., and Travers, A. (1992) DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen. EMBO J. 11, 2619–2625.

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13 In Vivo DNA Analysis Régen Drouin, Jean-Philippe Therrien, Martin Angers, and Stéphane Ouellet 1. Introduction The in vivo analysis of DNA–protein interactions and chromatin structure can provide several kinds of critical information regarding regulation of gene expression and gene function. For example, DNA sequences spanned by nuclease-hypersensitive sites or bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology it is possible to map such DNA sequences and to demonstrate the existence of unusual DNA structures directly in living cells. LMPCR analyses can thus be used as a primary investigative tool to identify the regulatory sequences involved in gene expression. Once specific promoter sequence sites are shown to be bound by transcription factors in living cells, it is often possible to establish the identity of these factors simply by comparison with the consensus binding sites of known factors such as Sp1, AP-1, NF-1, and so forth. The identity of each factor can then be confirmed using in vitro gel shift (electrophoretic mobility shift assay [EMSA]) or footprinting assays. Clearly, gene promoters are best studied in their natural state in the living cell and, thus, it is not surprising that in vivo DNA footprinting is one of the most accurate predictors of the state of transcriptional activity of genes (1–3). The native state of a gene and most of the special DNA structures are unavoidably lost when DNA is cloned or purified (1–4). Hence, the commonly used in vitro methods, such as in vitro footprinting and EMSAs, cannot demonstrate that a given DNA–protein interaction actually occurs within the cells of interest. With the advent of in vivo DNA footprinting, in vitro studies have been extended to the situation in living cells, revealing the cellular processes impliFrom: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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cated in the regulation of gene expression. LMPCR is the method of choice for in vivo footprinting and DNA structure studies because it can be used to investigate complex animal genomes, including that of human. The quality and usefulness of the information obtained from any in vivo DNA analysis, however, depends on three parameters: (1) the integrity of the native chromatin substrate used in the experiment, (2) the structural specificity of the chromatin probe, and (3) the sensitivity of the assay. The ideal chromatin substrate is, of course, that found inside intact cells. However, a near-ideal chromatin substrate is still to be found in permeabilized cells, allowing the application of a wider range of DNA cleavage agents, including DNase I. In vivo footprinting assesses the local reactivity of modifying agents on the DNA of living cells as compared to that on purified DNA (see Figs. 1–4). Two steps characterize an in vivo footprinting analysis: (1) the treatment of purified DNA and of cells with a given DNA modifying agent and (2) the visualization of nucleotide modifications on a DNA sequencing gel. The latter step requires that the modifying agent either directly induces DNA strand breaks or modifies DNA nucleotides such that strand breaks can subsequently be induced in vitro. A comparison is then made between the modification frequency on purified DNA and that on the DNA in living cells. For example, each guanine residue of purified DNA has a near-equivalent probability of being methylated by dimethylsulfate (DMS) and, thus, the cleavage pattern of in vitro modified DNA appears on a sequencing gel as a ladder of bands of roughly equal intensity. However, as a result of the presence of DNA-binding proteins, all guanine residues do not show the same accessibility to DMS in living cells (Fig. 1). Thus, differences between banding patterns obtained from in vitro and in vivo modified DNA can be used to infer the sites of protein binding in living cells. As will be seen, it is always advisable to validate such interpretations using more than one footprinting agent. The step of visualizing in vivo footprints has historically been problematic because of the dilute nature of the sequences of interest and the complexity of the genomes of higher eukaryotes. The development of an extremely sensitive and specific technique, such as LMPCR, was thus necessary. The LMPCR techFig. 1. (opposite page) Overall scheme for in vivo DNA analysis using DMS. The methylation of guanine residues following DMS treatment of purified DNA (in vitro) and cells (in vivo) is shown by vertical arrows and methylated residues (Me). When purified DNA is treated with DMS, every guanine residue has a similar probability of being methylated. However, the guanine residue in intimate contact with a sequencespecific DNA-binding protein illustrated by the dotted oval is protected from DMS methylation, whereas the guanine residues localized close to the boundary of a DNA– protein contact that modifies DNA structure, allowing a better accessibility to DMS, is


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methylated more frequently. The methylated guanine residues are cleaved by hot piperidine leaving phosphorylated 5' ends. On the sequencing ladder following LMPCR, guanine residues that are protected from methylation appear as missing or less intense bands when compared with the sequencing ladder from the same DNA sequence obtained after DMS treatment of purified DNA. On the other hand, guanine residues that undergo enhanced DMS methylation appear as darker bands in the sequencing ladder relative to the purified DNA control.

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Fig. 2. Overall scheme for in vivo DNA analysis using UVC and CPD formation. The CPD formation following UVC exposure of purified DNA (in vitro) and cells (in vivo) is shown with curved arrows and brackets linking two adjacent pyrimidines (Y). When purified DNA is irradiated with UVC, the frequency of CPD formation at dipyrimidine sites is determined by the DNA sequence. However, the presence of a sequence-specific DNA-binding protein illustrated by the dotted oval as well as DNA structure can prevent (negative photofootprint) or enhance (positive photofootprint)


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nique quantitatively maps single-strand DNA breaks having phosphorylated 5' ends within single-copy DNA sequences. It was first developed by Mueller and Wold (5) for DMS footprinting, and, subsequently, Pfeifer and colleagues adapted it to DNA sequencing (6), methylation analyses (1,6,7), DNase I footprinting (2), nucleosome positioning (2), and UV photofootprinting (4,8). LMPCR can be combined with a variety of DNA-modifying agents used to probe the chromatin structure in vivo. It is our opinion that no single technique can provide as much information on the DNA–protein interactions and DNA structures existing within living cells as can LMPCR.

1.1. General Overview of LMPCR Genomic sequencing techniques such as that developed by Church and Gilbert (9) can be used to map strand breaks in mammalian genes at nucleotide resolution. However, by incorporating an exponential amplification step, LMPCR (outlined in Fig. 5) constitutes a genomic sequencing method orders of magnitude more sensitive than the direct technique of Church and Gilbert. It uses 20 times less DNA than this latter technique to obtain a nucleotide-resolution banding pattern and allows short autoradiographic exposure times. The unique aspect of LMPCR is the blunt-end ligation of an asymmetric doublestranded linker (5' overhanging to avoid self-ligation or ligation in the wrong direction) onto the 5' end of each cleaved blunt-ended DNA molecule (5,6). The blunt end is created by the extension of a gene-specific primer (primer 1 in Fig. 5) until a footprinting strand break is reached. Because the generated breaks will be randomly distributed along the genomic DNA and thus have 5' ends of unknown sequence, the asymmetric linker adds a common and known sequence to all 5' ends. This then allows exponential PCR amplification from an adjacent genomic sequence to that of the generated breaks using the longer oligonucleotide of the linker (linker-primer) and a second nested gene-specific primer (primer 2, see Fig. 5). After 20–22 cycles of PCR, the DNA fragments are size-fractionated on a sequencing gel. LMPCR preserves the quantitative representation of each fragment in the original population of cleaved molecules (10–13), allowing quantification on a phosphorimager (14–17). Thus, the band intensity pattern obtained by LMPCR directly reflects the frequency distribu-

CPD formation. The CPDs are cleaved by T4 endonuclease V digestion and photolyase photoreactivation leaving phosphorylated 5' ends. On the sequencing ladder following LMPCR, the negative photofootprints appear as missing or less intense bands when compared with the sequencing ladder from the same DNA sequence obtained after UVC irradiation of purified DNA. On the other hand, positive photofootprints appear as darker bands in the sequencing ladder relative to the purified DNA control.

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Fig. 3. Overall scheme for in vivo DNA analysis using UVC and 6–4PP formation. The 6–4PP formation following UVC exposure of purified DNA (in vitro) and cells (in vivo) is shown with curved arrows and brackets linking two adjacent pyrimidines (Y). When purified DNA is irradiated with UVC, the frequency of 6–4PP formation at dipyrimidine sites is determined by the DNA sequence. However, the presence of a sequence-specific DNA-binding protein illustrated by the dotted oval as well as DNA structure can prevent (negative photofootprint) or enhance (positive photofootprint)


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tion of 5'-phosphoryl DNA breaks along a 200-bp sequence adjacent to the nested primer. Two methods exist to reveal the sequence and footprinting ladders created by LMPCR. Pfeifer and colleagues (6) took advantage of electroblotting DNA onto a nylon membrane followed by hybridization with a gene-specific probe to reveal sequence ladders, otherwise known as “indirect end labeling”. On the other hand, Mueller and Wold (5) used a nested third radiolabeled primer for the last one or two cycles of the PCR amplification step. We find Pfeifer’s method much more sensitive than Mueller and Wold’s (unpublished data). In this chapter, we will describe our LMPCR protocol as modified from the protocol of Pfeifer and colleagues. In summary, LMPCR is the method of choice to study the in vivo structure of promoters with respect to the positions of DNA–protein interactions, of special DNA structures and of chromatin structures such as nucleosomes. To perform in vivo DNA analysis, three probing agents are regularly combined with LMPCR: DMS, ultraviolet (UV) and DNase I (Figs. 1–4, Table 1). These probing agents provide complementary information and each has its associated advantages and drawbacks (Table 2). To best characterize DNA–protein interactions, it is often necessary to use two or even all three of these methods. Treatments with any probing agents must produce either strand breaks or modified nucleotides that can be converted to DNA strand breaks with a 5'-phosphate in vitro (Figs. 1–4, Table 3). In this chapter, we describe protocols routinely used in our laboratory for DMS, UV, and DNase I in vivo treatments as well as the associated LMPCR technology. These protocols may also be adapted to footprinting with other probing agents, such as KMnO4 and OsO4 (see Chapters 6 and 9), although a detailed description is beyond the scope of the present chapter.

1.2. In Vivo Dimethylsulfate (DMS) Footprint Analysis (Fig. 1) Dimethylsulfate is a small, highly reactive molecule that easily diffuses through the outer cell membrane and into the nucleus. It preferentially methylates not only the N7 position of guanine residues via the major groove but, to a lesser extent, also the N3 position of adenine residues via the minor groove. 6–4PP formation. First, CPDs are photoreactivated by photolyase and then 6–4PPs are cleaved by hot piperidine treatment leaving phosphorylated 5' ends. On the sequencing ladder following LMPCR, the negative photofootprints appear as missing or less intense bands when compared with the sequencing ladder from the same DNA sequence obtained after UVC irradiation of purified DNA. On the other hand, positive photofootprints appear as darker bands in the sequencing ladder relative to the purified DNA control.

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Fig. 4. Overall scheme for in vivo DNA analysis using DNase I. The DNase I enzyme (the solid black) digestion of purified DNA (in vitro) and cells (in vivo) is shown. When purified DNA is digested with DNase I, the cleavage pattern shows that sites of the nucleotide sequence have similar probabilities of being cleaved. However, the presence of a sequence-specific DNA-binding protein illustrated by the dotted oval


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The most significant technical advantage of in vivo DMS footprinting is that DMS can be simply added to the cell culture medium, requiring no cell manipulation (see Table 2 for advantages and drawbacks). Each guanine residue of purified DNA displays about the same probability of being methylated by DMS. Because DNA inside living cells forms chromatin and is often found associated with a number of proteins, it is expected that its reactivity toward DMS will differ from purified DNA. Figures 6 and 7 show in vivo DMS treatment patterns compared to the treatment of purified genomic DNA. Proteins in contact with DNA either decrease accessibility of specific guanines to DMS (protection) or, as frequently observed at the edges of a footprint, increase reactivity (hyperreactivity) (1). Hyperreactivity can also indicate a greater DMS accessibility of special in vivo DNA structures (19). Hot piperidine cleaves the glycosylic bond of methylated guanines and adenines, leaving a ligatable 5'-phosphate (20). Genomic footprinting using DMS reveals DNA–protein contacts located in the major groove of the DNA double helix (Table 1). However, it should be noted that in vivo DNA studies using DMS alone may not detect some DNA– protein interactions (21). First, no DNA–protein interaction will be detected in the absence of guanine residues. Second, some proteins do not affect DNA accessibility to DMS. Third, certain weak DNA–protein contacts could actually be disrupted because of the high reactivity of the DMS. Thus when using DMS, it is often important to also apply alternative footprinting approaches (21,22).

1.3. Photofootprint Analysis (Figs. 2 and 3) Ultraviolet light (UVC: 200–280 nm; UVB: 280–320 nm) can also be used as a modifying agent for in vivo footprinting (4,8,23–25). When cells are subjected to UV light (UVC or UVB), two major classes of lesions may be introduced into the DNA at dipyrimidine sequences (CT, TT, TC, and CC): the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6–4) pyrimidone photoproduct (6–4PP) (26). CPDs are formed between the 5,6 bonds of any two adjacent pyrimidines, whereas a stable bond between positions 6 and 4 of two adjacent pyrimidines characterizes 6–4PPs. 6–4PP are formed at a rate 15–30% of that of CPDs (27) and are largely converted to their Dewar valence as well as DNA structure can prevent (protection) or enhance (hypersensitive) DNase I cleavage. The DNase I cleavage leaves phosphorylated 5' ends. On the sequencing ladder following LMPCR, DNA sequences that are protected from DNase I cleavage appear as missing or less intense bands when compared with the sequencing ladder from the same DNA sequence obtained after DNase I digestion of purified DNA. On the other hand, hypersensitive sites that undergo enhanced DNase I cleavage appear as darker bands in the sequencing ladder relative to the purified DNA control.

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isomers by direct secondary photolysis (photoisomerization) (27). In living cells, the photoproduct distribution is determined both by sequence context and chromatin structure (28). In general, CPDs and 6–4PPs appear to form preferentially in longer pyrimidine runs. Because UVB and UVC radiation are primarily absorbed in the cell by the DNA, there are relatively few perturbations of other cellular processes, and secondary events that could modify the chromatin structure or release DNA–protein interactions. Furthermore, intact cells are exposed for a short period of time only to a high-intensity UV irradiation. Thus, UV irradiation is probably one of the least disruptive footprinting method and, hence, truly reflects the in vivo situation (Table 2). As for DMS, DNA-binding proteins influence the distribution of UV photoproducts in a significant way (23). When the photoproduct spectrum of irradiated purified DNA is compared with that obtained after irradiation of living cells, some striking differences become apparent. These are referred to as “photofootprints” (23). The photoproduct frequency within sequences bound by sequence-specific DNA-binding proteins (transcription factors) is suppressed or enhanced in comparison to purified DNA (4,8,29). Effects of chromatin structure may be significant in regulatory gene regions that bind transcription factors (Fig. 6). Mapping of CPDs at the single-copy gene level can reveal positioned nucleosomes because CPDs are modulated in a 10-bp periodicity within nucleosome core DNA (30,31). 6–4PPs form more frequently in linker DNA than in core DNA (32). Photofootprints reveal variations in DNA structure associated with the presence of transcription factors or other proteins bound to the DNA. UV light has the potential to reveal all DNA–protein interactions provided there is a dipyrimidine sequence on either DNA strand within a putative protein-binding sequence. Because photofootprints can be seen outside protein-binding sites, Fig. 5. (previous page) Outline of the LMPCR procedure. Step I: specific conversion of modified bases to phosphorylated 5' single-strand breaks; Step II: denaturation of genomic DNA; Step III: annealing and extension of primer 1 (although both strands can be studied, each LMPCR protocol only involves the analysis of either the nontranscribed strand or the transcribed strand); Step IV: ligation of the linker; Step V: first cycle of PCR amplification, this cycle is a linear amplification because only the gene-specific primer 2 can anneal; Step VI: cycles 2 to 22 of exponential PCR amplification of gene-specific fragments with primer 2 and the linker primer (the longer oligonucleotide of the linker); Step VII: separation of the DNA fragments on a sequencing gel, transfer of the sequence ladder to a nylon membrane by electroblotting, and visualization of the sequence ladder by hybridization with a labeled singlestranded probe; Step VIII: preparation and isotopic or nonisotopic labeling of singlestranded probe.

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Table 1 Purposes of the Three Main In Vivo Footprinting Approaches Approaches

Activities

1. Dimethylsulfate (DMS)

i. Localizes in vivo DNA–protein contacts located in the major groove of the DNA double helix ii. Can detect special DNA structures

2. UV irradiation (UVB or UVC)

i. Localizes in vivo DNA–protein interactions and shows how DNA structure is affected in the presence of transcription factors ii. Can detect special DNA structures iii. Can show evidence of positioned nucleosomes i. Localizes in vivo DNA–protein contacts ii. Precisely maps in vivo DNase I hypersensitive sites iii. Shows evidence of nucleosomes and their positions; can differentiate core DNA from linker DNA

3. DNase I

UV light should not be used as the only in vivo footprinting agent. The precise delimitations of the DNA–protein contact are difficult to determine with the simple in vivo UV probing method. The distribution of UV-induced CPDs and 6–4PPs along genomic DNA can be mapped at the sequence level by LMPCR following conversion of these photoproducts into ligatable 5'-phosphorylated single-strand breaks. CPD are enzymatically converted by cleavage with T4 endonuclease V followed by UVA (320–400 nm) photoreactivation of the overhanging pyrimidine using photolyase (Fig. 2) (8). Because the 6–4PPs and their Dewar isomers are hot alkali-labile sites, they can be cleaved by hot piperidine (Fig. 3) (29).

1.4. In Vivo DNase I Footprint Analysis (Fig. 4) DNase I treatment of permeabilized cells gives clear footprints when the DNase I-induced breaks are mapped by LMPCR (2). Both living cells (in vivo) and purified DNA (in vitro) are treated with DNase I. As with DMS and UV, footprint analyses are obtained by comparing in vivo DNase I digestion patterns to patterns obtained from the digestion of purified genomic DNA (Fig. 7). When compared to purified DNA, permeabilized cells show protected bands at DNA–protein interaction sequences and DNase I hypersensitive bands in regions of higher-order nucleoprotein structure (2). Compared to DMS, DNase I is less base selective, is more efficient at detecting minor groove DNA–protein contacts, provides more information on chromatin structure, displays larger and clearer footprints, and better delimits the boundaries of DNA– protein interactions (Fig. 7). The nucleotides covered by a protein are almost completely protected on both strands from DNase I nicking, allowing a better

Approaches DMS

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UV irradiation (UVB or UVC)

DNase I

Advantages Treatment is technically easy to carry out; the DMS is a small molecule that penetrates very easily into living cells with little disruption. 1. Treatment is technically easy to carry out; UV light penetrates through the outer membrane of living cells without disruption. 2. Detects many DNA–protein interactions. 3. Very sensitive to particular and DNA structures. 1. Little sequence dependency. 2. No conversion of modified bases required. 3. Detects all DNA-protein contacts. 4. Very sensitive to particular DNA structures.

Drawbacks


Table 2 Advantages and Drawbacks of the Three Main In Vivo Footprinting Approaches

1. Requires guanines, therefore is sequence dependent. 2. Does not detect all DNA–protein interactions. 1. Requires two adjacent pyrimidines, therefore is sequence dependent. 2. The interpretation of the results is sometime difficult; to differentiate between DNA–protein interactions. and special DNA structures can be very difficult. 1. Technically difficult to carry out; reproducibility is often a problem. 2. DNase I is a protein that can penetrate in living cells only following membrane permeabilization, thus causing some cell disruption.

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Table 3 Mapping Schemes Used with the Three Main In Vivo Footprinting Approaches

Approaches

Strand breaks

DMS

Few

UV irradiation (UVB or UVC)

Very few

DNase I

Yes

Modified bases Guanine: methylated guanines at N7 position Adenine: to a much lesser extent, methylated adenines at N3 position (i) Cyclobutane pyrimidine dimers (ii) 6–4 Photoproducts None

Conversion of modified bases to DNA single-strand breaks Hot piperidine

(i) T4 endonuclease V followed by photolyase (ii) Photolyase followed by hot piperidine No conversion required

delimitation of the boundaries of DNA–protein contacts. However, it should be underlined that the relatively bulky DNase I molecule cannot cleave the DNA in the immediate vicinity of a bound protein because of steric hindrance. Consequently, the regions protected from cutting can extend beyond the actual DNA–protein contact site. On the other hand, when DNA is wrapped around a nucleosome-size particle, DNase I cutting activity is increased at 10-bp intervals and no footprint is observed (Tables 1 and 2). DNase I, a relatively large 31-kDa protein, cannot penetrate cells without previous cell-membrane permeabilization. Cells can be efficiently permeabilized by lysolecithin (2) or Nonidet P40 (33). It has been shown that cells permeabilized by lysolecithin remain intact, replicate their DNA very efficiently, and show normal transcriptional activities (34,35). There are numerous studies showing that lysolecithin-permeabilized cells maintain a normal nuclear structure to a greater extent than isolated nuclei, because the chromatin structure can be significantly altered during the nuclear isolation procedures (2). Indeed, DNase I footprinting studies using isolated nuclei can be flawed because transcription factors are lost during the isolation of nuclei in polyamine containing buffers (2). Even though other buffers may be less disruptive, factors can still be lost during the isolation procedure, leading to the loss of footprints or partial loss of footprints. DNase I digestion of DNA leaves ligatable 5’-phosphorylated breaks, but the 3’-ends are free hydroxyl groups. Pfeifer and colleagues (2,36) observed that these genomic 3'-OH ends can be used as primers and be extended by the DNA polymerases during the initial extension and/or PCR steps of LMPCR, thereby reducing significantly the overall efficiency of LMPCR and giving a


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Fig. 6. LMPCR analysis of methylated guanines and CPD along the nontranscribed strand of the c-jun promoter following DMS treatment, and UVB and UVC irradiation respectively. (A) The membrane was hybridized with an isotopic [32P]-dCTP-labeled probe. The membrane was exposed on film between two intensifying screens for 25 min at –70°C. (B) The membrane was hybridized with a digoxigenin-labeled probe and exposed on film for 40 min at room temperature. For this experiment, one LMPCR protocol was carried out and only one gel was run on which all the samples (20 in total) were loaded symmetrically in duplicate. Each symmetrical well of each set of samples was loaded with exactly the same amount of DNA. Lanes 1–4: LMPCR of DNA-treated with chemical cleavage reactions. These lanes represent the sequence of the c-jun promoter analyzed with JD primer set (18). Lanes 5–6: LMPCR of DMStreated naked DNA (T: in vitro) and fibroblasts (V: in vivo) followed by hot piperidine treatment. Lanes 7–10: LMPCR of UVC- and UVB-irradiated naked DNA (T) and fibroblasts (V) followed by T4 endonuclease V/photolyase digestion. On the right, the consensus sequences of transcription factor binding sites are delimited by brackets. The numbers indicate their positions relative to the major transcription initiation site.

background smear on sequencing gels. To avoid the nonspecific priming of these 3'-OH ends, three alternative solutions have been applied: (1) blocking these ends by the addition of a dideoxynucleotide (2,36); (2) enrichment of fragments of interest by extension product capture using biotinylated genespecific primers and magnetic streptavidin-coated beads (18,37–39); and (3) performing primer 1 hybridization and primer 1 extension at a higher temperature (52–60°C vs 48°C, and 75°C vs 48°C, respectively) using a thermostable

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Fig. 7. LMPCR analysis of methylated guanines and DNA strand breaks along the transcribed strand of the c-jun promoter following DMS treatment and DNase I digestion, respectively. The membrane was hybridized with an isotopic [32P]-dCTP-labeled probe. Lanes 1–2: LMPCR of DMS-treated purified DNA (t: in vitro) and fibroblasts (v: in vivo) followed by hot piperidine treatment. Lanes 3–6: LMPCR of DNA-treated with chemical cleavage reactions. These lanes represent the sequence of the c-jun promoter analyzed with JC primer set (18). Lanes 7–8: LMPCR of DNase I-digested permeabilized fibroblasts (v) and purified DNA (t). As a reference, a small portion of the chemically derived sequence is shown on the right of the autoradiogram, the AP-1-like binding sequence is enclosed by a box, and the numbers indicate its position relative to the major transcription initiation site. Open circles represent guanines


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enzyme such as Vent exo– DNA polymerase and cloned Pfu DNA polymerase (3,40–42). Although effective, the first two alternatives involve additional manipulations that are time-consuming. Because of its simplicity, we select primer 1 with a higher Tm (52–60°C) and use the cloned Pfu DNA polymerase for the primer 1 extension.

1.5. Choice of DNA Polymerases for LMPCR Ligation-mediated PCR involves the PCR amplification of a mixture of genomic DNA fragments of different size. During the LMPCR procedure, DNA polymerases are required for two steps: primer extension (PE) and PCR amplification. For the PE step, the best DNA polymerase would be one that (1) is thermostable and very efficient, (2) has no terminal transferase activity, (3) is able to efficiently polymerize about 0.75 kb of DNA even when the DNA is very GC rich, and (4) is able to polymerize through any DNA secondary structures. For the PCR step, the best DNA polymerase would be (1) thermostable, (2) very efficient, (3) able to amplify indiscriminately a mixture of DNA fragments of different lengths (between 50 and 750 bp) and of varying GC-richness (from 5% to 95%), and (4) able to efficiently resolve DNA secondary structures. We find cloned Pfu DNA polymerase that corresponds to Pfu exo– is the best enzyme for the PE and PCR steps of LMPCR (42). In this chapter, LMPCR protocols using cloned Pfu DNA polymerase for PE and PCR steps will be described in detail. However, because the more frequently used combination of DNA polymerases is Sequenase™ 2.0 for the PE step and Taq DNA polymerase for the PCR step, a description of an alternative LMPCR protocol using Sequenase 2.0 and Taq DNA polymerase will also be included. 2. Materials 2.1. DNA Purification (for 107 to 108 cells) 1. 2. 3. 4.

Any types of cells (i.e., fibroblasts, lymphocytes, etc.). Trypsin–EDTA (Gibco-BRL). Hank’s Balanced Salt Solution (HBSS) (Gibco-BRL). Buffer A: 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 60 mM Tris-HCl, pH 8.0, 0.5 mM spermidine, 0.15 mM spermine, and 2 mM EDTA. Store at –20°C. 5. Buffer A + 1% Nonidet P40. Store at –20°C. 6. Conical tissue culture tubes, 50 mL. 7. Buffer B: 150 mM NaCl and 5 mM EDTA pH 7.8.

that are protected against DMS-induced methylation (negative DMS footprints) in vivo. The black bar shows the protected sequence against DNase I-induced cleavage in vivo. Thus, in vivo DNase I footprinting analysis delimits much better the DNA– protein interactions.

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8. Buffer C: 20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 20 mM EDTA, and 1% sodium dodecyl sulfate (SDS). 9. Proteinase K from Tritirachium album (Roche Molecular Biochemicals). 10. RNase A from bovine pancreas (Roche Molecular Biochemicals). 11. Phenol, equilibrated with 0.1 M Tris-HCl, pH 8.0 (Roche Molecular Biochemicals, cat. no. 108-95-2). 12. Chloroform. 13. 5 M NaCl. 14. Precooled absolute ethanol (–20°C). 15. Precooled 70% ethanol (–20°C). 16. N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). 17. 4'–6-Diamidino-2-phenylindole (DAPI). 18. Nanopure H2O should be used in making any buffers, solutions, and dilutions, unless otherwise specified.

2.2. Chemical Cleavage for DNA-Sequencing Products 1. Potassium tetrachloropalladate(II) (K2PdCl4, Aldrich). 2. K2PdCl4 solution: 10 mM K2PdCl4 and 100 mM HCl, pH 2.0 (adjusted with NaOH). Store at –20°C. 3. K2PdCl4 stop: 1.5 M sodium acetate, pH 7.0, and 1 M β-mercaptoethanol. 4. Dimethylsulfate (DMS, 99+%, Fluka). Considering its toxic and carcinogenic nature, DMS should be manipulated in a well-ventilated hood. DMS is stored under nitrogen at 4°C and should be replaced every 12 mo. DMS waste is detoxified in 5 M NaOH. 5. DMS buffer: 50 mM sodium cacodylate and 1 mM EDTA pH 8.0. Store at 4°C. 6. DMS stop: 1.5 M sodium acetate pH 7.0 and 1 M β-mercaptoethanol. Store at –20°C. 7. Hydrazine (Hz, anhydrous, Aldrich). Considering its toxic and carcinogenic potentials, Hz should be manipulated in a well-ventilated hood. Hz is stored under nitrogen at 4°C in an explosion-proof refrigerator and the bottle should be replaced at least every 6 mo. Hz waste is detoxified in 3 M ferric chloride. 8. Hz stop: 300 mM sodium acetate pH 7.0 and 0.1 mM EDTA. Store at 4°C. 9. 5 M NaCl. 10. 3 M Sodium acetate pH 7.0. 11. Precooled absolute ethanol (–20°C). 12. Precooled 80% ethanol (–20°C). 13. Dry ice. 14. Piperidine (99+%, Fluka or Sigma): 10 M stock diluted to 2 M with H2O just before use by adding 250 µL stock under 1 mL H2O in a 1.5-mL microtube on ice. Cap immediately to minimize evaporation. Considering its toxic and carcinogenic potentials, piperidine should be manipulated in a well-ventilated hood. Piperidine 10 M is stored at 4°C under nitrogen atmosphere. 15. Teflon tape. 16. Lock caps. 17. 3 M Sodium acetate pH 5.2. 18. 20 µg/µL glycogen. 19. Vacuum concentrator (SpeedVac concentrator, Savant).


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2.3. Treatment of Purified DNA and Living Cells with Modifying Agents 2.3.1. DMS Treatment 1. DMS (99+%, Fluka). 2. Trypsin–EDTA (Gibco-BRL). 3. Hank’s Balanced Salt Solution (HBSS).

2.3.2. 254-nm UV and UVB Irradiation 1. 2. 3. 4. 5.

Germicidal lamp (254 nm) for UVC irradiation (Philips G15 T8, TUV 15W). UVB light for UVB irradiation (Philips, FS20T12/UVB/BP). UVX digital radiometer (Ultraviolet Products, Upland, CA). 0.9% NaCl. UV irradiation buffer: 150 mM KCl, 10 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. 6. Buffer A + 0.5% Nonidet P40. Store at –20°C. 7. Scraper.

2.3.3. DNase I Treatment 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Deoxyribonuclease I (DNase I, Worthington biochemical corporation; 45A134). Trypsin–EDTA (Gibco-BRL). Hank’s Balanced Salt Solution (HBSS). L -α-Lysophosphatidylcholine (L-α-Lysolecithin). Nonidet P40. Solution I: 150 mM sucrose, 80 mM KCl, 35 mM HEPES, pH 7.4, 5 mM MgCl2, and 0.5 mM CaCl2. Solution II: 150 mM sucrose, 80 mM KCl, 35 mM HEPES, pH 7.4, 5 mM MgCl2, and 2 mM CaCl2. Conical tubes, 15 and 50 mL. Buffer B: 150 mM NaCl and 5 mM EDTA, pH 7.8. Buffer C: 20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 20 mM EDTA, and 1% SDS. Proteinase K from Tritirachium album (Roche Molecular Biochemicals). RNase A from bovine pancreas (Roche Molecular Biochemicals). Phenol (see Subheading 2.1., item 11). Chloroform. 5 M NaCl. Precooled absolute ethanol (–20°C). Precooled 80% ethanol (–20°C).

2.4. Conversion of Modified Bases to DNA Single-Strand Breaks 2.4.1. DMS-Induced Base Modifications Piperidine (99+%, see Subheading 2.2., item 14).

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2.4.2. UV-Induced Base Modifications 1. 10X dual buffer: 500 mM Tris-HCl, pH 7.6, 500 mM NaCl, and 10 mM EDTA. 2. 1 M 1,4-Dithiothreitol (DTT, Roche Molecular Biochemicals). 3. 5 mg/mL nuclease-free bovine serum albumine (BSA, Roche Molecular Biochemicals). 4. T4 endonuclease V enzyme (Epicentre Technologies). The saturating amount of T4 endonuclease V enzyme can be estimated by digesting UV-irradiated genomic DNA with various enzyme quantities and separating the cleavage products on alkaline agarose gel (43). The saturating amount of the enzyme is the next to the minimum quantity that produces the maximum cleavage frequency as evaluated on the alkaline agarose gel. 5. E. coli photolyase enzyme (Pharmingen). The saturating amount of photolyase can be estimated by photoreactivating UV-irradiated genomic DNA with various enzyme quantities, digestion with T4 endonuclease V, and separating the cleavage products on alkaline agarose gel (43). The saturating amount of photolyase is the next to the minimum enzyme quantity which produces no cleavage following T4 endonuclease V digestion as evaluated on the gel. Because photolyase is light sensitive, all steps involving photolyase should be carried out under yellow light. 6. UVA black light (UV F15T8BLB 360 nm, Philips, 15W). 7. Plastic film (plastic wrap). 8. 0.52% SDS solution. 9. Phenol (see Subheading 2.1., item 11). 10. Chloroform. 11. 5 M NaCl. 12. Precooled absolute ethanol (–20°C). 13. Precooled 80% ethanol (–20°C). 14. Piperidine (99+%, see Subheading 2.2., item 14).

2.5. Ligation-Mediated Polymerase Chain Reaction Technology 2.5.1. Primer Extension (Steps II and III, Fig. 5) 1. A gene-specific primer (primer 1) is used to initiate primer extension. The primer 1 used in the first-strand synthesis are 15- to 22-mer oligonucleotides and have a calculated melting temperature (Tm) of 50–60°C. They are selected using a computer program (Oligo 4.0 software, National Biosciences) (44) and, optimally, their Tm, as calculated by a computer program (GeneJockey software), should be about 10°C lower than that of subsequent primers (see Note 1) (45). The firststrand synthesis reaction is designed to require very little primer 1 with a lower Tm so that this primer does not interfere with subsequent steps (11–13,46). The primer 1 concentration is set at 50 µM in H2O and then diluted 1:100 in H2O to give 0.5 pmol/µL. 2. Siliconized microtubes (0.625 µL) (National Scientific Supply Co, Inc.). 3. Thermocycler (PTC™, MJ research, Inc.).


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4. 10X cloned Pfu buffer: 200 mM Tris-HCl, pH 8.8, 20 mM MgSO4, 100 mM NaCl, 100 mM (NH4)2SO4, 1% (v/v) Triton X-100, and 1 mg/mL nuclease-free BSA (see Note 2). 5. Cloned Pfu mix: 1.5 mM of each dNTP and 1.5 U cloned Pfu DNA polymerase, also named Pfu exo– (2.5 U/µL, Stratagene). 6. 5X Sequenase buffer: 200 mM Tris-HCl, pH 7.7, and 250 mM NaCl. 7. Mg–dNTPs mix: 20 mM MgCl2, 20 mM DTT, and 0.375 mM of each dNTP. 8. T7 Sequenase V.2 (Amersham). 9. 310 mM Tris-HCl, pH 7.7.

2.5.2. Ligation (Step IV, Fig. 5) 1. The DNA molecules that have a 5' phosphate group and a double stranded blunt end are suitable for ligation. A DNA linker with a single blunt end is ligated directionally onto the double-stranded blunt end of the extension product using T4 DNA ligase. This linker has no 5' phosphate and is staggered to avoid selfligation and provide directionality. Also, the duplex between the 25-mer (5' GCGGTGACCCGGGAGATCTGAATTC) and 11-mer (5' GAATTCAGATC) is stable at the ligation temperature, but denatures easily during subsequent PCR reactions (5,46). The linker was prepared in aliquots of 500 µL by annealing in 250 mM Tris-HCl, pH 7.7, 20 pmol/µL each of the 25-mer and 11-mer, heating at 95°C for 3 min, transferring quickly at 70°C, and cooling gradually to 4°C over a period of 3 h. Linkers are stored at –20°C and thawed on ice before use. Linker: L25 (60 pmol/µL, 5'-GCGGTGACCCGGGAGATCTGAATTC), L11 (60 pmol/µL, 5'-GAATTCAGATC), 2 M Tris-HCl, pH 7.7, and 1 M MgCl2. 2. T4 DNA ligase (1 U/µL, Roche Molecular Biochemicals). 3. Ligation mix: 30 mM DTT, 1 mM ATP, 83.3 µg/mL of BSA, 100 pmol of linker, and 3.25 U/microtube of T 4 DNA ligase. If cloned Pfu DNA polymerase was used for primer extension (step III, Fig. 5), the ligation mix is prepared by adding per microtube: 1.35 µL of 1 M DTT, 0.5 µL of 100 mM ATP, 0.15 µL of 5 µg/µL BSA, 1.1 µL of Tris-HCl, pH 7.4, 5.0 µL of 20 pmol/µL linker, 3.25 µL of 1 U/µL T4 ligase, and 33.65 µL of H2 O. If Sequenase was used for primer extension (step III, Fig. 5), the ligation mix is prepared by adding per microtube: 1.35 µL of 1 M DTT, 0.5 µL of 100 mM ATP, 0.75 µL of 5 µg/µL BSA, 5.0 µL of 20 pmol/µL linker, 3.25 µL of 1 U/µL T4 ligase, and 34.15 µL of H2O. 4. 7.5 M ammonium acetate. 5. 0.5 M EDTA, pH 8.0. 6. 20 µg/µL glycogen. 7. Precooled absolute ethanol (–20°C). 8. Precooled 80% ethanol (–20°C).

2.5.3. Polymerase Chain Reaction (Steps V and VI, Fig. 5) 1. At this step, gene-specific fragments can be exponentially amplified because primer sites are available at each end of the target fragments (i.e., primer 2 on one end and the longer oligonucleotide of the linker on the other end). Primer 2 may

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5. 6. 7.

8. 9. 10.

11. 12. 13. 14.

Drouin et al. or may not overlap with primer 1. The overlap, if present, should not be more than seven to eight bases (11–13,46). Primer 2 is diluted in H2O to give 50 pmol/µL. 10X cloned Pfu buffer: 200 mM Tris-HCl, pH 8.8, 20 mM MgSO4, 100 mM NaCl, 100 mM (NH4)2SO4, 1% (v/v) Triton X–100, and 1 mg/mL nuclease-free BSA (see Note 2). Cloned Pfu DNA polymerase, also named Pfu exo– (2.5 U/µL, Stratagene). Cloned Pfu DNA polymerase mix per microtube: 2X cloned Pfu buffer, 0.5 mM of each dNTP, 10 pmol of LP25 (Linker Primer), 10 pmol of primer 2, and 3.5 U of cloned Pfu DNA polymerase. Mineral oil. Cloned Pfu DNA polymerase stop: 1.56 M sodium acetate pH 5.2 and 20 mM EDTA. Formamide loading dye: 94% formamide, 2 mM EDTA, pH 7.7, 0.05% xylene cyanole FF, and 0.05% bromophenol blue (11–13). The formamide loading dye is freshly premixed by adding 1 part H2O to 2 parts formamide loading dye. 5X Taq buffer: 50 mM Tris-HCl, pH 8.9, 200 mM NaCl, and 0.05% [w/v] gelatin (see Note 2). Taq DNA polymerase (5 U/µL, Roche Molecular Biochemicals). Taq DNA polymerase mix per microtube: 2X Taq buffer, 4 mM MgCl2, 0.5 mM of each dNTP, 10 pmol LP25 (Linker Primer), 10 pmol primer 2, and 3 U Taq DNA polymerase. Taq DNA polymerase stop: 1.56 M sodium acetate pH 5.2 and 60 mM EDTA. Phenol (see Subheading 2.1., item 11) premixed with chloroform in a ratio of 92 µL of phenol for 158 µL of chloroform. Precooled absolute ethanol (–20°C). Precooled 80% ethanol (–20°C).

2.5.4. Gel Electrophoresis and Electroblotting (Step VII, Fig. 5) 60-cm-long × 34.5-cm-wide sequencing gel apparatus (Owl Scientific). Spacers (0.4-mm thick). Plastic well-forming comb (0.4-mm thick, Bio-Rad). 5X (0.5 M) Tris–borate–EDTA (TBE) buffer: 500 mM Tris, 830 mM boric acid, and 10 mM EDTA, pH 8.3. Use this stock to prepare 1X (100 mM) TBE buffer. 5. 8% Polyacrylamide, to prepare 1 L, add 77.3 g of acrylamide, 2.7 g of bisacrylamide, 420.42 g of urea, and 200 mL of 0.5 M TBE dissolved in H2O. Polyacrylamide solution should be kept at 4°C. 6. Gel preparation: Mix 100 mL of 8% polyacrylamide with 1 mL of 10% ammonium persulfate (APS) and 30 µL of N,N,N',N'-tetra-methylethylenediamide (TEMED). This mix is prepared immediately before pouring the solution between the glass plates. Without delay, take the gel mix into a 50-mL syringe and inject the mix between the plates, maintaining a steady flow. During pouring, the plates should be kept at a 30° angle and tilted to the side into which the mix is injected. Any air bubbles should be avoided and removed if they form. The gel should be left to polymerize for a minimum of 3 h before use. If the gel is to be left over-

1. 2. 3. 4.


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night, 45 min after pouring, place a moistened paper tissue over the comb, and cover the upper end of the assembly with a plastic film to prevent the gel from drying out. Flat gel loading tips (National Scientific Supply Co). Power supply (Bio-Rad PowerPac 3000). Electroblotting apparatus (HEP3, Owl Scientific Inc.) used according to the manufacturer’s instructions. Whatman 3MM Chr paper (Fisher Scientific). Plastic film (plastic wrap). Whatman 17 Chr papers (Fisher Scientific). Nylon membrane, positively charged (Roche Molecular Biochemicals, cat. no. 1 417 240). Power supply (Bio-Rad, model 200/2.0). UVC (254 nm) germicidal lamp.

2.5.5. Hybridization (Step VII, Fig. 5) The hybridization is performed in a rolling 8-cm-diameter × 22 cm long borosilicate glass hybridization tubes in a hybridization oven (Hoefer). The nylon membrane is soaked in 100 mM TBE and, using a 25-mL pipet, placed in the tube so that the membrane sticks completely to the wall of the hybridization tube. Following hybridization and washing, the membranes are placed in an autoradiography cassette FBAC 1417 (Fisher Scientific) and exposed to Kodak X-ray film (XAR-5, 35 × 43 cm, Kodak Scientific Imaging Film) with intensifying screens (35 × 43 cm, Fisher Scientific, cat. no. FB-IS-1417) at –70°C when a radiolabeled probe has been hybridized and without intensifying screens at room temperature when a digoxigenin-labeled probe has been hybridized. 2.5.5.1. RADIOLABELED PROBE 1. Hybridization buffer: 250 mM sodium phosphate pH 7.2, 1 mM EDTA, 7% SDS, and 1% BSA. 2. Radiolabeled probe diluted in 6–7 mL of hybridization buffer. 3. Washing buffer I: 20 mM sodium phosphate pH 7.2, 1 mM EDTA, 0.25% BSA, and 2.5% SDS. 4. Washing buffer II: 20 mM sodium phosphate pH 7.2, 1 mM EDTA, and 1% SDS. 5. Plastic film (plastic wrap).

2.5.5.2. DIGOXIGENIN-LABELED PROBE 1. Prehybridization buffer: 5X SSC (750 mM NaCl and 75 mM sodium citrate pH 7.0), 1% casein, 0.1% N-lauroylsarcosin, and 0.02% SDS. 2. Digoxigenin-labeled probe diluted in 15 mL of prehybridization buffer (use only 7.5 mL for hybridization). 3. 2X washing solution: 2X SSC and 0.1% SDS.

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4. 5. 6. 7. 8. 9. 10.

0.5X washing solution: 0.5X SSC and 0.1% SDS. Buffer 1: 150 mM NaCl and 100 mM maleic acid, pH 7.5. Buffer 2: buffer 1 + 1% (w/v) casein. Antidigoxigenin antibodies (Roche Molecular Biochemicals). Buffer 1 + 0.3% Tween-20. Buffer 3: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2. CSPD® [disodium 3-(4-methoxyspiro {1,2-dioxetane–3,2'-(5' chloro)tricyclo [3.3.1.13,7]decan}–4-yl)phenyl phosphate] substrate (Roche Molecular Biochemicals, cat. no. 1 655 884). 11. Acetate sheets. 12. Doubleseal (Model 855, Decosonic).

2.6. Preparation of Single-Stranded Hybridization Probes (Step VIII, Fig. 5) 2.6.1. Template Preparation: PCR Products 2.6.1.1. PCR AMPLIFICATION 1. 5X Taq buffer: 50 mM Tris-HCl, pH 8.9, 200 mM NaCl, and 0.05% (w/v) gelatin (see Note 2). 2. Taq DNA polymerase (5 U/µL, Roche Molecular Biochemicals). 3. One primer 2 (50 pmol/µL) for each strand of the DNA fragment to be amplified distant from 150 to 450 bp. 4. Taq DNA polymerase mix per microtube: 2X Taq buffer, 4 mM MgCl2, 0.4 mM of each dNTP, 10 pmol of each primer 2, and 3 U Taq DNA polymerase. 5. Mineral oil. 6. Taq DNA polymerase stop: 1.56 M sodium acetate, pH 5.2, and 60 mM EDTA. 7. Phenol (see Subheading 2.1., item 11) premixed with chloroform in a ratio of 92 µL of phenol to 158 µL of chloroform. 8. Precooled absolute ethanol (–20°C). 9. Precooled 80% ethanol (–20°C). 10. 5X TAE loading buffer: 5X TAE (200 mM Tris base, 100 mM glacial acetic acid, and 5 mM EDTA, pH 8.0), 0.025% bromophenol blue, 30% Ficoll 400, and 2% SDS.

2.6.1.2. PURIFICATION AND QUANTIFICATION OF PCR PRODUCTS 1. Agarose. 2. 1X TAE buffer: 40 mM Tris base, 20 mM glacial acetic acid, and 1 mM EDTA, pH 8.0. 3. DNA size standards (φX 174 RF, Canadian life technologies, cat. no. 15611-015). 4. Ethidium bromide. 5. Siliconized microtubes (0.625 mL) and 1.5-mL microtubes. 6. Glass wool. 7. 3 M sodium acetate, pH 7.0. 8. Precooled absolute ethanol (–20°C).


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9. Precooled 80% ethanol (–20°C). 10. Low DNA mass ladder (Gibco-BRL, cat. no. 10068-013). 11. 5X universal neutral loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol in H2O. Store at 4°C.

2.6.2. Labeling of Single-Strand Hybridization Probes 2.6.2.1. ISOTOPIC LABELING 1. Siliconized microtubes (0.625 mL) and 1.5-mL microtubes. 2. 5X Taq buffer: 50 mM Tris-HCl, pH 8.9, 200 mM NaCl, and 0.05% (w/v) gelatin (see Note 2). 3. 100 mM MgCl2. 4. DNA templates: PCR products (10 ng/µL) or DNA plasmids (20 ng/µL). 5. Primer 2 (50 pmol/µL). 6. dNTP (dATP, dGTP, dTTP) mix (200 µM of each). 7. dNTP (dATP, dGTP, dTTP) mix diluted 1:10 in H2O. This mix is changed every 2 wk. 8. Taq DNA polymerase (5 U/µL, Roche Molecular Biochemicals). 9. α-[32P]dCTP (3000 Ci/mmol, New England Nuclear). 10. 7.5 M ammonium acetate. 11. 20 µg/µL glycogen 12. Precooled absolute ethanol (–20°C) 13. Geiger counter. 14. TE buffer pH 8.0: 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, pH 7.8. 15. Hybridization buffer: 250 mM sodium phosphate, pH 7.2, 1 mM EDTA, 7% SDS, and 1% BSA.

2.6.2.2. DIGOXIGENIN (NONISOTOPIC) LABELING 1. Siliconized microtubes (0.625 mL) and 1.5-mL microtubes. 2. 5X Taq buffer: 50 mM Tris-HCl, pH 8.9, 200 mM NaCl, and 0.05% (w/v) gelatin (see Note 2). 3. 100 mM MgCl2. 4. DNA templates: PCR products (10 ng/µL) or DNA plasmids (20 ng/µL). 5. Primer 2 (50 pmol/µL). 6. dNTP mix (A:G:C:T = 25 mM : 25 mM : 25 mM : 20 mM). 7. dNTP mix diluted 1:8.3 in H2O. This mix is changed every 2 wk. 8. 1 mM digoxigenin-11-dUTP (Roche Molecular Biochemicals) diluted 1:2 in H2O. 9. Taq DNA polymerase (5 U/µL, Roche Molecular Biochemicals). 10. 7.5 M ammonium acetate. 11. 20 µg/µL glycogen. 12. Precooled absolute ethanol (–20°C). 13. TE buffer pH 8.0: 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, pH 7.8. 14. Prehybridization buffer: 5X SSC (750 mM NaCl and 75 mM sodium citrate pH 7.0), 1% casein, 0.1% N-lauroylsarcosine, and 0.02% SDS.

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3. Methods 3.1. DNA Purification (for 107 to 108 Cells) 1. Detach cells using trypsin (if needed) and sediment the cell suspension by centrifugation in 50-mL conical tubes. 2. Resuspend the cells in 5–15 mL of buffer A. 3. Add 1 volume (5–15 mL) of buffer A containing 1% Nonidet P40. 4. Incubate on ice for 5 min. 5. Sediment nuclei by centrifugation at 4500g for 10 min at 4°C. 6. Remove the supernatant. Resuspend nuclei in 1–10 mL of buffer A by gentle vortexing. Resediment nuclei at 4500g for 10 min at 4°C. 7. Remove supernatant. It is recommended to leave a small volume (100–500 µL) of buffer A to facilitate resuspension of nuclei. 8. Dilute the nuclei in 1–2 mL of buffer B. 9. Add an equivalent volume of buffer C and proteinase K to a final concentration of 450 µg/mL. 10. Incubate at 37°C for 3 h, shake occasionally (see Note 3). 11. Add RNase A to a final concentration of 150 µg/mL. 12. Incubate at 37°C for 1 h. 13. Purify DNA by extraction with 1 vol phenol (one to two times as needed), 1 vol phenol:chloroform (one to two times as needed), and 1 vol chloroform. Phenol extraction and phenol-chloroform extraction should be repeated if the aqueous phase is not clear (see Note 3). 14. Precipitate DNA in 200 mM NaCl and 2 vol of precooled absolute ethanol. Ethanol should be added slowly and to facilitate DNA recovery, rock the tube very gently. 15. Recover DNA by spooling the floating DNA filament with a micropipet tip. If DNA is in small pieces or not clearly visible, recover DNA by centrifugation (5000g for 30 min at 4°C), but expect RNA contamination (see Note 4). RNA contamination does not cause any problems for LMPCR. RNase digestion can be repeated if needed. 16. Remove supernatant and wash DNA once with 10 mL of 70% ethanol. 17. Centrifuge the DNA (5000g for 10 min at 4°C). 18. Remove supernatant and air-dry DNA pellet. 19. Dissolve DNA in 10 mM HEPES, pH 7.4, and 1 mM EDTA (HE buffer) at an estimated concentration of 60–100 µg/mL. The quantity of DNA can be estimated based upon the number of cells that were initially used for DNA purification. About 6 µg of DNA should be purified from 1 × 106 cells. 20. Carefully measure DNA concentration by spectrophotometry at 260 nm. Alternatively, DNA can be measure by fluorometry after staining with DAPI. Only double-strand DNA concentration has to be measured, be careful if there is RNA contamination (see Note 5).

3.2. Chemical Cleavage for DNA Sequencing Products In vivo DNA analysis using LMPCR requires complete DNA sequencing ladders from genomic DNA. Base-specific chemical modifications are per-


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formed according to Iverson and Dervan (47) for the A reaction and Maxam and Gilbert for the G, T+C, and C reactions. DNA from each of these basemodification reactions is processed by LMPCR concomitantly with the analyzed samples and loaded in adjacent lanes on the sequencing gel to allow the identification of the precise location and sequence context of footprinted regions. The chemical modifications induced by DMS, Hz, and K2PdCl4 and cleaved by piperidine destroy the target base. Therefore, one must bear in mind that when analyzing a chemical-sequencing ladder, each band corresponds to a DNA fragment ending at the base preceding the one read. In this section, we will describe the chemical sequencing of genomic DNA. The cleavage protocol below works optimally with 10–50 µg of genomic DNA per microtube. Before chemical sequencing, the required amount of DNA per microtube is ethanol precipitated and the pellet is air-dried. For each base-specific reaction, we usually carried out the treatment in three microtubes containing 50 µg of genomic DNA for three different incubation times with the modifying agent in order to obtain low, medium and high base-modification frequencies.

3.2.1. A Reaction 1. Add 160 µL of H2O and 40 µL of K2PdCl4 solution to the DNA pellet and carefully mix on ice using a micropipet. 2. Incubate at 20°C for 5, 10, or 15 min. 3. Add 50 µL of K2PdCl4 stop. 4. Add 750 µL of precooled absolute ethanol.

3.2.2. G Reaction 1. Add 5 µL of H2O, 200 µL of DMS buffer, and 1 µL of DMS to the DNA pellet and carefully mix on ice using a micropipet. 2. Incubate at 20°C for 30, 45, or 60 s. 3. Add 50 µL of DMS stop. 4. Add 750 µL of precooled absolute ethanol.

3.2.3. T+C Reaction 1. Add 20 µL of H2O and 30 µL of Hz to the DNA pellet and carefully mix on ice using a micropipet. 2. Incubate at 20°C for 120, 210, or 300 s. 3. Add 200 µL of Hz stop. 4. Add 750 µL of precooled absolute ethanol.

3.2.4. C Reaction 1. Add 5 µL of H2O, 15 µL of 5 M NaCl, and 30 µL of Hz to the DNA pellet and carefully mix on ice using a micropipet. 2. Incubate at 20°C for 120, 210, or 300 s.

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3. Add 200 µL of Hz stop. 4. Add 750 µL of precooled absolute ethanol. All samples are processed as follows: 1. Mix samples well and place on dry ice for 15 min. 2. Centrifuge for 15 min at 15,000g at 4°C. 3. Remove supernatant, then recentrifuge for 1 min and remove all the liquid using a micropipet. 4. Carefully dissolve pellet in 405 µL of H2O. 5. Add 45 µL of 3 M sodium acetate pH 7.0. 6. Add 1 mL of precooled absolute ethanol. 7. Leave on dry ice for 15 min. 8. Centrifuge for 15 min at 15,000g at 4°C. 9. Take out supernatant and then respin. 10. Wash with 1 mL of precooled 80% ethanol; spin 5 min at 15,000g in a centrifuge at 4˚C. 11. Remove the supernatant, spin quickly, remove the liquid with a micropipet and air-dry pellet. 12. Dissolve pellet in 50 µL of H2O, add 50 µL of freshly prepared 2 M piperidine, and mix well using a micropipet. 13. Secure caps with Teflon™ tapes and lock the caps with “lock caps”. 14. Incubate at 82°C for 30 min. 15. Pool all three microtubes of the same chemical reaction in a new 1.5-mL microtube. 16. Add H2O until a volume of 405 µL is reached, then add 10 µL of 3 M sodium acetate pH 5.2, 1 µL of glycogen, and 1 mL of precooled absolute ethanol. 17. Leave on dry ice for 15 min. 18. Spin 10 min at 15,000g at 4°C. 19. Take out the supernatant and wash twice with 1 mL of precooled 80% ethanol, then respin for 1 min and remove all the liquid using a micropipet. 20. Add 200 µL of H2O and remove traces of remaining piperidine by drying the sample overnight in a Speedvac concentrator. 21. Dissolve DNA in H2O to a concentration of 0.5 µg/µL. 22. Determine the DNA strand break frequency by running the samples on a 1.5% alkaline agarose gel (43). The size range of the fragments should span 100– 500 bp.

3.3. Treatment of Purified DNA and Cells with Modifying Agents 3.3.1. DMS Treatment 1. If cells are grown to confluence as monolayer, replace the cell culture medium with a freshly prepared serum-free medium containing 0.2% DMS and incubate at room temperature for 6 min. If cells are grown in suspension, sediment the cells by centrifugation and remove the cell culture medium. The cells are diluted


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in a freshly prepared serum-free medium containing 0.2% DMS and are then incubated at room temperature for 6 min. Remove the DMS-containing medium and quickly wash the cell monolayer with 10–20 mL of cold HBSS. Sediment cells by centrifugation if they are treated in suspension and remove the DMS-containing medium and wash the cells with 10 mL of cold HBSS. Detach cells using trypsin for cells grown as monolayer. Nuclei are isolated and DNA purified as described in Subheading 3.1. Purified DNA obtained from the same cell type is treated as described in Subheading 3.2.2. Usually, a DMS treatment of 45 s should give a break frequency corresponding to that of the in vivo treatment described in this section. This DNA is the in vitro treated DNA used to compare with DNA DMS-modified in vivo (see Notes 5 and 6).

3.3.2. 254-nm UV and UVB Irradiation 1. If cells are grown as monolayer in Petri dishes, replace cell culture medium with cold 0.9% NaCl. If cells are grown in suspension, sediment the cells by centrifugation and remove the cell culture medium. The cells are diluted in cold 0.9% NaCl at a concentration of 1 × 106 cells/mL (see Note 7) and, to avoid cellular shielding, a thin layer of the cell suspension is placed in 150-mm Petri dishes. 2. Expose the cells to 0.5–2 kJ/m2 of UVC (254-nm UV) or 25–100 kJ/m2 of UVB. The cells should be exposed on ice in uncovered Petri dishes. The UV intensity is measured using a UVX digital radiometer. 3. Remove the 0.9% NaCl by aspiration for cells grown as monolayer in Petri dishes or by sedimentation for cell suspensions. 4. If cells were irradiated in suspension; follow the procedure described in Subheading 3.1. to isolate nuclei and purify DNA. After DNA purification, DNA is dissolved in H2O at a concentration of 0.2 µg/µL. For cells cultured in Petri dishes, add in each dish 8 mL of buffer A containing 0.5% Nonidet P40. 5. Incubate on ice for 5 min. 6. Scrape the cells and transfer them in a conical 50-mL tube. In the same conical 50-mL tube, pool cells from Petri dishes that undergo the same procedure. 7. Wash the dishes twice with 8 mL of buffer A + 0.5% Nonidet P40 per each of three identical Petri dishes. 8. Continue from step 5 of Subheading 3.1. After DNA purification, DNA is dissolved in H2O at a concentration of 0.2 µg/µL. 9. Expose purified DNA to the same UVC or UVB dose as the cells. Purified DNA should be irradiated on ice and diluted in the UV irradiation buffer at a concentration of 60–75 µg/mL (see Note 6). Purified DNA should be obtained from the same type of cells as the type irradiated in vivo (see Note 8). This DNA is used as control DNA to compare with DNA UV modified in vivo (see Notes 6 and 7). 10. Following UV irradiation, DNA is ethanol precipitated and DNA is resuspended in H2O at a concentration of 0.2 µg/µL.

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3.3.3. DNase I Treatment Genomic footprinting with DNase I requires cell permeabilization (see Note 9). Cells grown as a monolayer can be permeabilized while they are still attached to the Petri dish or in suspension following trypsinization. Here, we will describe cell permeabilization using lysolecithin applied to monolayer cell cultures (steps labeled a). For monolayer cultures, cells are grown to about 80% of confluency. Alternatively, we describe cell permeabilization using lysolecithin or Nonidet P40 applied to cells in suspension (steps labeled b). For cells in suspension, cells are diluted at a concentration of approx 1 × 106 cells/mL. To permeabilize the vast majority of cells in suspension, they must not be clumped and not form aggregates during the permeabilization step and subsequent DNase I treatment. To achieve this, we gently flick the microtubes during permeabilization and DNase I treatment and keep the cell concentration below 2 × 106/µL. 1a. For cells in monolayers, permeabilize the cells by treating them with 4 mL of 0.05% lysolecithin in solution I (prewarmed) at 37°C for 1–2 min (48). 2a. Remove the lysolecithin and wash with 10 mL of solution I. Add 3 mL of DNase I (2–4 U/mL) to solution II and incubate at room temperature for 3–5 min. DNase I concentration and incubation times may have to be adjusted for different cell types. During this incubation, no more than 10% of the cells should be released from the dish. 3a. Stop the reaction and lyse the cells by removal of the DNase I solution and addition of 1.5 mL of buffer C containing 600 µg/mL of proteinase K. Add 1.5 mL of buffer B and mix gently by rocking the flask or the Petri dish. Transfer lysis solution to a 15-mL tube (then continue to step 4).

Alternatively: 1b. Sediment the cell suspension by centrifugation. Wash the cells with HBSS. Resuspend the cells in solution II at a concentration of 20 × 106/mL and aliquote by transferring 100 µL of the cell suspension per 1.5-mL microtube. Add to each microtube 100 µL of solution II prewarmed at 37°C containing 0.1% lysolecithin or 0.25% Nonidet P40. Mix gently by flicking. Incubate at room temperature for 3 min. 2b. Quickly spin to pellet the cells. Add 50 µL of 2000 U/mL DNase I and mix gently by flicking. Incubate at room temperature for 5 min. 3b. Quickly spin and remove supernatant, resuspend the cells in 1.5 mL of buffer B, and, using a pipet, rapidly transfer to a 15-mL tube in which there are and 1.5 mL of buffer C containing 600 µg/mL of proteinase K (then continue to step 4). 4. 5. 6. 7.

Incubate at 37°C for 3 h, shake occasionally. Add RNase A to a final concentration of 200 µg/mL and incubate at 37°C for 1 h. Purify DNA by phenol-chloroform extraction (see Subheading 3.1., step 13). Precipitate DNA in 200 mM NaCl and 2 volumes of precooled absolute ethanol.


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8. Leave on dry ice for 20 min. 9. Recover DNA by centrifugation (5000g for 30 min at 4°C), but expect RNA contamination. RNA contamination does not cause any problems for LMPCR. RNase A digestion can be repeated if needed. 10. Remove supernatant and wash DNA once with 10 mL of precooled 80% ethanol. 11. Centrifuge the DNA (5000g for 10 min at 4°C). Remove supernatant and air-dry DNA pellet. 12. Dissolve DNA in H2O and carefully measure DNA concentration (see Subheading 3.1., step 20). 13. To obtain purified DNA controls (see Notes 6 and 8), digest 50 µg of purified DNA in solution II with 4–8 U/µL of DNase I at room temperature for 10 min. Stop the reaction by adding 400 µL of phenol. Extract once with phenol–chloroform and once with chloroform. Dissolve DNA in H2O at a concentration of 0.5 µg/µL.

3.4. Conversion of Modified Bases to DNA Single-Strand Breaks When purified DNA or cells are treated with DMS and UV, DNA base modifications are induced (Table 3). These modifications must be converted to single-strand breaks before running LMPCR. Following UV exposure, CPDs and 6–4PPs are converted individually because they use different conversion procedures (Table 3). On the other hand, DNase I digestion generates DNA strand breaks suitable for LMPCR without any conversion procedures. Before running LMPCR, the DNA strand break frequency must be determined by running the samples on a 1.5% alkaline agarose gel (43). The size range of the fragments should span 200–2000 bp (see Note 6).

3.4.1. DMS-Induced Base Modifications (see Fig. 1) 1. Dissolve DNA (10–50 µg) in 50 µL H2O, add 50 µL of 2 M piperidine and mix well using a micropipet. 2. Samples are processed as described in Subheading 3.2., steps 13–20. 3. Dissolve DNA in H2O to a concentration of 0.2 µg/µL.

3.4.2. UV-Induced Base Modifications 3.4.2.1. CPD (SEE FIG. 2) 1. To specifically cleave CPDs, dissolve 10 µg of UV-irradiated DNA in 50 µL H2O, add 50 µL of a solution containing 10 µL of 10X dual buffer, 0.1 µL of 1 M DTT, 0.2 µL of 5 mg/mL BSA, a saturating amount of T4 endonuclease V and complete with H2O to a final volume of 50 µL. Mix well by flicking the microtube and quick spin. 2. Incubate at 37°C for 1 h. 3. To perform the photolyase digestion to remove the overhanging dimerized base that would otherwise prevent ligation (8), add 10 µL of the following mix: 1 µL of 10X dual buffer, 1 µL of 1 M DTT, 0.2 µL of 5 mg/mL BSA, a saturating amount of photolyase, and complete with H2O to a final volume of 10 µL. Mix well by flicking the microtube and quick spin.

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4. Preincubate the microtubes at room temperature for 3–5 min under yellow light with their caps opened. 5. Leaving their caps open, cover the microtubes with a plastic film to prevent UVBinduced damage and place open ends 2–3 cm from a UVA black light for 1 h. 6. Add 290 µL of 0.52% SDS, mix well, and extract DNA using 1 vol (400 µL) phenol, 1 vol phenol:chloroform, and 1 volume chloroform. 7. To precipitate DNA, add 18 µL of 5 M NaCl and 1 mL of precooled absolute ethanol. 8. Leave 15 min on dry ice, spin 20 min at 15,000g in a centrifuge at 4°C. 9. Wash once with 1 mL of precooled 80% ethanol. 10. Spin 8 min at 15,000g in a centrifuge at 4°C. 11. Air-dry the pellet and dissolve DNA in H2O to a concentration of 0.2 µg/µL.

3.4.2.2. 6–4PP (SEE FIG. 3) 1. Dissolve DNA (10–50 µg) in 50 µL of H2O, add 50 µL of 2 M piperidine and mix well using a micropipet. 2. Samples are processed as described in Subheading 3.2., steps 13–20. 3. Dissolve DNA in H2O to a concentration of 0.2 µg/µL.

3.5. Ligation-Mediated Polymerase Chain Reaction Technology The LMPCR protocol using cloned Pfu DNA polymerase for primer extension and PCR steps is labeled with a in Subheadings 3.5.1. and 3.5.3. An alternative LMPCR protocol using Sequenase for primer extension steps and Taq DNA polymerase for PCR steps is labeled b in Subheadings 3.5.1. and 3.5.3. Aside from the ligation mix (see Subheading 2.5.2.), the ligation step (Subheading 3.5.2.) is identical with both enzyme combinations. The primer extension, ligation, and PCR steps are carried out in siliconized 0.625-mL microtubes and a thermocycler is used for all incubations.

3.5.1. Primer Extension (Steps II and III, Fig. 5) 1a. Mix 0.5–2 µg of genomic DNA, 3 µL of cloned Pfu buffer, and 1 pmol of primer 1 in a final volume of 25 µL. 2a. Denature DNA at 98°C for 3 min. 3a. Incubate for 20 min at 45°C to 55°C, depending of the Tm of the primer 1. 4a. Cool to 4°C. 5a. Add 5 µL of the cloned Pfu mix. Flick and quick spin. 6a. Incubate the samples at the annealing temperature for 30 s, then increase the temperature to 75°C at a rate of 0.3°C/s and incubate at 75°C for 10 min. Finally, the samples are cooled to 4°C.

Alternatively: 1b. Mix 0.5–1.6 µg of DNA in Sequenase buffer with 1 pmol of primer 1 in a final volume of 15–18 µL. 2b. Denature DNA at 98°C for 3 min.


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3b. Incubate for 20 min at 45°C to 50°C, depending of the Tm of the primer 1. 4b. Cool to 4°C. 5b. Add 9 µL of the following mix: 7.5 µL of Mg–dNTP mix, 1.1 µL of H2O, and 0.4 µL of T7 Sequenase V.2. Flick and quick spin. 6b. Incubate at 48°C for 5 min, 50°C for 1 min, 51°C for 1 min, 52°C for 1 min, 54°C for 1 min, 56°C for 1 min, 58°C for 1 min, and 60°C for 1 min. Then, the samples are cooled to 4°C. 7b. Add 6 µL of cold 310 mM Tris-HCl, pH 7.7. 8b. Incubate at 67°C for 15 min to inactivate the Sequenase, then cool to 4°C.

3.5.2. Ligation (Step IV, Fig. 5) 1. To the primer extension reaction, add 45 µL of the ligation mix and mix well with the pipet. Note that the composition of the ligation mix (see Subheading 2.5.2.) is different whether Sequenase or cloned Pfu DNA polymerase was used for the primer extension (Section 3.5.1 and Step III in Fig. 5). 2. Incubate at 18°C overnight. 3. On ice, precipitate DNA by adding 28.75 µL of 7.5 M ammonium acetate, 0.25 µL of 0.5 M EDTA, pH 8.0, 1 µL of 20 µg/µL glycogen, and 275 µL of precooled absolute ethanol. 4. Leave 15 min on dry ice and spin 20 min at 15,000g in a centrifuge at 4°C. 5. Wash once with 500 µL of precooled 80% ethanol. 6. Spin 8 min at 15,000g in a centrifuge at 4°C. 7. Air-dry DNA pellets and dissolve DNA pellets in 50 µL of H2O.

3.5.3. Polymerase Chain Reaction (Steps V and VI, Fig. 5) 1a. Add 50 µL of the cloned Pfu DNA polymerase mix and mix with a pipet. The reaction mix is overlaid with 50 µL of mineral oil. 2a. Cycle 22 times as described in Table 4 for cloned Pfu DNA polymerase. The last extension should be done for 10 min to fully extend all DNA fragments. 3a. Add 25 µL of cloned Pfu DNA polymerase stop under the mineral oil layer. Then, continue to step 4.

Alternatively: 1b. Add 50 µL of the Taq DNA polymerase mix and mix with the pipet. The reaction is overlaid with 50 µL of mineral oil. 2b. Cycle 22 times as described in Table 4 for Taq DNA polymerase. The last extension should be done for 10 min to fully extend all DNA fragments. 3b. Add 25 µL of Taq DNA polymerase stop mix under the mineral oil layer. Then, continue to step 4. 4. Extract with 250 µL of premixed phenol–chloroform (92 µL :158 µL) and transfer to 1.5-mL microtubes. 5. Add 400 µL of precooled absolute ethanol. 6. Leave 15 min on dry ice; spin 20 min at 15,000g in a centrifuge at 4˚C.

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Table 4 Exponential Amplification Steps Using Cloned Pfu DNA Polymerase or Taq DNA Polymerase

Denaturation T in °C for D in s) Cycle 0 1 2 3 4 5

Annealing (T is the Tm of the oligonucleotide for D in s)

Polymerization (D in s) T is the same for all cycles: 75°C for Pfu and 74°C for Taq

Pfu

Taq

Pfu or Taq

—

— 98 for 300 98 for 120 98 for 60 98 for 30 98 for 20

93 for 120 98 for 150 95 for 60 95 for 60 95 for 60 95 for 60

— Tm for 180 Tm –1°C for 150 Tm –2°C for 120 Tm –3°C for 120 Tm –4°C for 90

— 180 180 180 180 150

Repeat cycle 5, 13 more times (add 5 s per cycle for annealing and polymerization) 19 20 21 22

98 for 20 98 for 20 98 for 20 98 for 20

95 for 60 95 for 60 95 for 60 95 for 60

Tm –3°C for 240 Tm –2°C for 240 Tm –1°C for 240 Tm for 240

240 240 240 600

Note: Temperature (T) and duration (D) of the denaturation, annealing and polymerization steps.

7. 8. 9. 10.

Wash once with 500 µL of precooled 80% ethanol. Spin 8 min at 15,000g in a centrifuge at 4°C. Air-dry DNA pellets. Dissolve DNA pellets in 7.5 µL of premixed formamide loading dye in preparation for sequencing gel electrophoresis. For the sequence samples G, A, T+C, and C, it is often advisable to dissolve DNA pellets in 15 µL of premixed formamide loading dye.

3.5.4. Gel Electrophoresis and Electroblotting (Step VII, Fig. 5) The PCR-amplified fragments are separated by electrophoresis through a 8% polyacrylamide/7 M urea gel, 0.4 mm thick and 60–65 cm long, then transferred to a nylon membrane by electroblotting (11–13). 1. Prerun the 8% polyacrylamide gel until the temperature of the gel reaches 50°C. Running buffer is 100 mM TBE. Before loading the samples, wash the wells thoroughly using a syringe. 2. To denature DNA, heat the samples at 95°C for 2–3 min, then keep them on ice prior to loading. 3. Load an aliquot of 3–3.5 µL using flat tips. 4. Run the gel at the voltage or power necessary to maintain the temperature of the gel at 50°C. This will ensure that the DNA remains denatured.


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5. Stop the gel when the green dye (xylene cyanole FF) reaches 1–2 cm from the bottom of the gel. 6. Separate the glass plates using a spatula, then remove one of the plates by lifting it carefully. The gel should stick to the less treated plate (see Note 10). 7. Cover the lower part of the gel (approx 40–42 cm) with a clean Whatman 3MM Chr paper, carefully remove the gel from the glass plate and cover it with a plastic film (see Note 10). 8. On the bottom plate of the electroblotter, individually layer three sheets of Whatman 17 CHR paper, 43 cm × 19 cm, presoaked in 100 mM TBE, and squeeze out the air bubbles between the paper layers by rolling with a bottle or pipet. 9. Add 150 mL of 100 mM TBE on the top layer and place the gel quickly on the Whatman 17 CHR papers before TBE is absorbed. Before removing the plastic film, remove all air bubbles under the gel by gentle rolling with a 10-mL pipet. 10. Remove the plastic film and cover the gel with a positively charged nylon membrane presoaked in 100 mM TBE, remove all air bubbles by gently rolling a 10-mL pipet, then cover with three layers of presoaked Whatman 17 CHR paper and squeeze out air bubbles with rolling bottle. Paper sheets can be reused several times except for those immediately under and above the gel. 11. Place the upper electrode onto the paper. 12. Electrotransfer for 45 min at 2 A. The voltage should settle at approximately 10–15 V. 13. UV-crosslink (1000 J/m2 of UVC) the blotted DNA to the membrane, taking care to expose the DNA side of the membrane. If probe stripping and rehybridization are planned, keep the membrane damp.

3.5.5. Hybridization (Step VII, Fig. 5) 3.5.5.1. RADIOLABELED PROBE 1. Prehybridize with 15 mL of hybridization buffer at 60–68°C for 20 min. The prehybridization temperature is based on the Tm of the primer used to prepare the probe. 2. Decant the prehybridization buffer and add the labeled probe in 6–8 mL of hybridization buffer. 3. Hybridize at 60–68°C (2°C below the calculated Tm of the probe) overnight. 4. Wash the membrane with prewarmed washing buffers. The buffers should be kept in an incubator or water bath set at a temperature of 4°C higher than the hybridization temperature. The membrane is placed into a tray on an orbital shaker. Wash with buffer I for 10 min and with buffer II three times for about 10 min each time. 5. Wrap the membrane in plastic film (see Note 10). Do not let the membrane become dry if stripping and rehybridization are planned after exposure to the film. 6. Expose membrane to X-ray films with intensifying screens at –70°C. Although longer exposure might be necessary, an exposure of 0.5–8 h is usually enough to produce a sharp autoradiogram. Nylon membranes can be rehybridized if more than one set of primers have been included in the primer extension and amplification reactions (11–13). Probes can be stripped by soaking the membranes in boiling 0.1% SDS solution twice for 5–10 min each time.

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3.5.5.2. DIGOXIGENIN-LABELED PROBE 1. Prehybridize with 20 mL of prehybridization buffer at 60–68°C for at least 3 h. 2. Decant the prehybridization buffer and add 7.5 mL of digoxigenin-labeled probe in prehybridization buffer. 3. Hybridize at 60–68°C (2°C below the calculated Tm of the probe) overnight. 4. Wash the membrane twice with 20 mL of 2X washing solution for 5 min each at room temperature, followed by two washes with 20 mL of 0.1X washing solution for 15 min each at 65°C. The membrane is placed into a rolling 8-cm-diameter × 22-cm-long borosilicate glass hybridization tube in a hybridization oven. Manipulate the membrane exclusively with tweezers (see Note 11) and do not let it dry following the hybridization step. 5. Wash the membrane with 50 mL of buffer 1 for 1 min at room temperature. 6. Transfer the membrane to a new hybridization tube and incubate with 20 mL of buffer 2 for 1 h at room temperature. 7. Replace buffer 2 with 20 mL of buffer 2 containing the antidigoxigenin antibody diluted 1:10,000 (prepared 5 min before use) and incubate for 30 min at room temperature. 8. Remove the antibody solution and wash the membrane with 20 mL of buffer 1. 9. Transfer the membrane to a new hybridization tube and incubate with 20 mL of buffer 1 containing 0.3% Tween-20 for 15 min at room temperature. 10. Replace the solution with 20 mL of buffer 3 and incubate for 5 min at room temperature. 11. Place the membrane between two cellulose acetate sheets and pour 0.5 mL:100 cm2 of CSPD® diluted 1:100 in buffer 3 onto the membrane between the acetate sheet sandwich. Carefully remove the air bubbles and seal the acetate sheets using heat (Doubleseal). Incubate the membrane for 15 min at 37°C. 12. Expose membrane to X-ray films for 40 min at room temperature (see Note 11).

3.6. Preparation of Single-Stranded Hybridization Probes (Step VIII, Fig. 5) The [32P]-dCTP or digoxigenin-labeled single-stranded probe is prepared by 30 cycles of repeated linear primer extension using Taq DNA polymerase. Primer 2 (or primer 3, see Note 12) is extended on a double-stranded template which can be a plasmid or a PCR product. The latter is produced using two opposing primers 2, separated by a distance of 150–450 bp. Alternatively, any pair of gene specific primers suitable for amplifying a DNA fragment containing a suitable probe sequence (see Note 12) can be employed.

3.6.1. Template Preparation: PCR Products 3.6.1.1. PCR AMPLIFICATION 1. To 50 µL of purified genomic DNA (100 ng) in H2O, add 50 µL of the Taq DNA polymerase mix and mix with the pipet. The reaction is overlaid with 50 µL of mineral oil.


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2. Cycle 35 times at 95°C for 1 min (97°C for 3 min for the first cycle), 61–73°C (1–2°C below the calculated Tm of primer 2 with the lowest Tm) for 2 min, and 74°C for 3 min. The last extension should be done for 10 min. 3. Add 25 µL of Taq DNA polymerase stop under the mineral oil layer. 4. Extract with 250 µL of premixed phenol–chloroform (92 µL:158 µL) and transfer to 1.5-mL microtubes. 5. Add 400 µL of precooled absolute ethanol. 6. Leave 15 min on dry ice, spin 20 min at 15,000g in a centrifuge at 4°C. 7. Wash once with 1 mL of precooled 80% ethanol. 8. Spin 8 min at 15,000g in a centrifuge at 4°C. 9. Air-dry DNA pellets. 10. Resuspend DNA pellets in 25 µL of 1X TAE loading buffer.

3.6.1.2. PURIFICATION AND QUANTIFICATION OF PCR PRODUCTS 1. Load 25 µL of PCR products per well along with an appropriate DNA mass ladder. 2. Migrate the PCR products on a neutral 1.2–1.5% agarose gel. 3. Stain the gel with ethidium bromide and recover the band containing the DNA fragment of expected molecular weight on a UV transilluminator using a clean scalpel blade. Minimize the size of the slice by removing as much extraneous agarose as possible. 4. Crush the slice and put it in a 0.625-mL microtube pierced at the bottom and containing a column of packed dry glass wool (see Note 13). 5. Insert the 0.625-mL microtube containing the column in a 1.5-mL microtube and spin 15 min at 7000g. Transfer the flowthrough to a new 1.5-mL microtube. If there is still some visible agarose remaining, repeat step 5. 6. Add 50 µL of H2O to wash the column of any remaining DNA by spinning 8 min at 7000g. Pool all of the flowthrough contents in one 1.5-mL microtube. 7. Complete the volume to 405 µL with H2O, add 45 µL of 3 M sodium acetate pH 7.0, and 1 mL of precooled absolute ethanol to precipitate DNA. Leave 15 min on dry ice, spin 20 min at 15,000g in a centrifuge at 4°C. 8. Wash once with 1 mL of precooled 80% ethanol and spin 8 min at 15,000g in a centrifuge at 4°C. 9. Air-dry DNA pellet. 10. Dissolve DNA pellets in 103 µL of H2O. 11. Load aliquots of 1 and 2 µL of the DNA template dissolved in 1X universal neutral loading buffer along with a quantitative DNA molecular-weight ladder and electrophorese on a neutral 1.5% agarose gel. 12. Stain the gel with ethidium bromide and photograph on a UV transilluminator. The DNA concentration of the aliquots is estimated by comparison with the DNA ladder band intensities and H2O is added to obtain a final concentration of template DNA of 3 ng/µL. The DNA template is aliquoted and stored at –20˚C.

3.6.2. Labeling of Single-Strand Hybridization Probes 3.6.2.1. ISOTOPIC LABELING 1. Prepare 150 µL of the following mix: 30 µL of 5X Taq buffer, 3 µL of 100 mM MgCl2, 1 µL of dNTPs mix diluted 1:10 in H2O, 20–40 ng of plasmid or 10–20 ng

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2. 3. 4. 5. 6.

7. 8.

Drouin et al. of PCR products, 1.5 µL of 50 pmol/µL primer 2, 2.5 U of Taq DNA polymerase, and 10 µL of α-[32P]-dCTP (3000 ci/mmol). Cycle 30 times at 95°C for 1 min (97°C for 3 min for the first cycle), 60–68°C for 2 min, and 74°C for 3 min. Transfer the mixture to a conical 1.5-mL microtube with screw cap. Precipitate the probe by adding 50 µL of 10 M ammonium acetate, 1 µL of glycogen, and 420 µL of precooled absolute ethanol. Leave 5 min at room temperature and spin 5 min at 15,000g in a centrifuge at room temperature. Transfer the supernatant to into a new 1.5-mL microtube. Using a Geiger counter, compare the counts per minute between the pellet (probe) and the supernatant, count from the probe should be more than or equal to the supernatant for optimal results. Dissolve the probe in 100 µL of TE buffer. Add the probe to 6–8 mL of hybridization buffer and keep the probe at 65°C.

3.6.2.2. DIGOXIGENIN (NONISOTOPIC) LABELING 1. Prepare 150 µL of the following mix: 30 µL of 5X Taq buffer, 3 µL of 100 mM MgCl2, 1 µL of dNTP mix diluted 1:8.3, 20–40 ng of plasmid or 10–20 ng of PCR products, 1.5 µL of 50 pmol/µL primer 2, 2.5 U of Taq DNA polymerase, and 1.2 µL of 0.5 mM digoxigenin–11-dUTP. 2. Cycle 30 times at 95°C for 1 min (97°C for 3 min for the first cycle), 60–68°C for 2 min, and 74°C for 3 min. 3. Precipitate the probe by adding 50 µL of 10 M ammonium acetate, 1 µL of glycogen, and 420 µL of precooled absolute ethanol. Spin for 10 min at 15,000g in a centrifuge at room temperature. 4. Check the incorporation of the digoxigenin-labeled nucleotide by a dot blot (see Note 14). 5. Resuspend the probe in 100 µL of TE buffer. 6. Add the probe to 15 mL of prehybridization buffer.

4. Notes 1. Primers should be selected to have a higher Tm at the 5' end than in the 3' end. This higher annealing capacity of the 5' end lowers false priming, thus allowing a more specific extension and less background (49). A guanine or a cytosine residue should also occur at the 3' end. This stabilizes the annealing and facilitates the initiation of the primer extension. It is important that the selected primer does not have long runs of purines or pyrimidines, does not form loops or secondary structure, and does not anneal with itself. If primer dimerization occurs, less primer will be available for annealing and polymerization will not be optimal. The purity of the primers is verified on a 20% polyacrylamide gel (to prepare a 500-mL mix, dissolve 96.625 g of acrylamide, 3.375 g of bis-acrylamide, 210.21 g of urea corresponding to 7 M, in 100 mM TBE); if more than one band is found, the primer is reordered. The primers are also tested in a conventional PCR to prepare the template for the probe synthesis (see Note 12).


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2. Originally, Pfu and Taq buffers were prepared using KCl, which was, however, shown to stabilize secondary DNA structures, thus preventing an optimal polymerization (50). The use of NaCl prevents, to some extent, the ability of DNA to form secondary structures. This is particularly helpful when GC-rich regions of the genome are being investigated. 3. The genomic DNA used for LMPCR needs to be very clean and undegraded. Any shearing of the DNA during preparation and handling before the first primer extension must be avoided. After an incubation of 3 h, if clumps of nuclei are still visible, proteinase K at a final concentration of 300 µg/mL should be added and the sample reincubated at 37˚C for another 3 h. 4. If no DNA can be seen, add glycogen (1–2 µg) to the DNA solution and put the DNA on dry ice for 20 min and centrifuge the DNA (5000g for 20 min at 4°C). This should help DNA recovery but increases the probability of RNA contamination. 5. Because in vivo DNA analysis is based on the comparison of DNA samples modified in vivo with DNA control modified in vitro, given the quantitative characteristic and high sensitivity of LMPCR technology, the DNA concentrations should be as accurate as possible. Indeed, it is critical to start LMPCR with similar amounts of DNA in every sample to be analyzed. The method used to evaluate DNA concentration should measure only double-stranded nucleic acids. RNA contamination does not affect LMPCR, although it can interfere with the precise measurement of the DNA concentration. 6. The DNA frequency of DNA breakage is even more critical than the DNA concentration. For DMS and UV, the base-modification frequency determines the break frequency following conversion of the modified bases to single-strand breaks, whereas for DNase I, the frequency of cleavage is exactly the break frequency. The break frequency must be similar among the samples to be analyzed. It should not average more than one break per 150 bp for in vivo DNA analysis, the optimal break frequency varying from one break per 200 bp to one break per 2000 bp. When the break frequency is too high, we typically observe dark bands over the bottom half of the autoradiogram and very pale bands over the upper half, reflecting the low number of long DNA fragments. In summary, to make the comparison of the in vivo modified DNA sample with a purified DNA control easily interpretable and valid, the amount of DNA and the break frequency must be similar between the samples to be compared. On the other hand, it is not so critical that the break frequency of the sequence ladders (G, A, T+C, and C) be similar to that of the samples to be studied. However, to facilitate sequence reading, the break frequency should be similar between the sequence reactions. It is often necessary to load less DNA for the sequence ladders. 7. If the cell density is too high, multiple cell layers will be formed and the upper cell layer will obstruct the lower ones. This will result in an inhomogeneous DNA photoproduct frequency. 8. It is imperative that the purified DNA samples used as DNA control and the in vivo DNA samples come from the same cell type. For instance, differing cytosine

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methylation patterns of genomic DNA from different cell types affect photoproduct formation (17,29) and give altered DNase I cleavage patterns (2). 9. A nearly ideal chromatin substrate can be maintained in permeabilized cells. Nonionic detergents such as lysolecithin (48) and Nonidet P40 (33) permeabilize the cell membrane sufficiently to allow the entry of DNase I. Conveniently, this assay can be performed with cells either in a suspension or in a monolayer. One concern is that permeabilized cells will lyse after a certain time in a detergent, thus care must be taken to monitor cell integrity by microscopy during the course of the experiment. A further difficulty with the permeabilization technique concerns the relatively narrow detergent concentration range over which the treatment can be performed. Each cell type appears to require specific conditions for the detergent cell permeabilization. Furthermore, the DNase I concentration must be calibrated for each cell type to produce an appropriate cleavage frequency. Optimally, the in vivo DNase I protocol works better if the enzyme has cleaved the DNA backbone every 1.5–2 kb. Cutting frequencies greater than 1 kb are associated with higher LMPCR backgrounds because the number of 3'-OH ends is much higher, making suppression of the extension of these ends more difficult. 10. To facilitate sequencing gel removal following migration, it is crucial to siliconize the inner face of both glass plates prior to pouring the gel. For security, costeffectiveness, efficiency, and time-saving, we recommend treating the glass plates with RAIN-AWAY® solution (Wynn’s Canada, product no. 63020). We apply 0.75 mL on one plate and 1.5 mL on the other before each five utilizations as specified by the manufacturer. In this way, the gel is easier to pour and tends to stick on the less siliconized plate. Whenever plastic film is needed, we recommend plastic wrap brand. This brand was found to be less permeable to liquids and more resistant to tears than other brands. This is particularly important when membranes are exposed on the phosphorimager plate in order to avoid the moistening of the plate and irreversibly damaging it. 11. We adapted the nonisotopic digoxigenin-based probe labeling method and chemiluminescent detection system (Roche Molecular Biochemicals) to reveal DNA sequence ladders after LMPCR amplification, sequencing gel electrophoresis, and electroblotting. Compared to the isotopic method, the nonisotopic method has a higher specificity, higher sensitivity, lower background, and lower cost, and is therefore a highly recommendable alternative. As shown in Fig. 6B, the sequence ladder revealed by nonisotopic labeling was clearer, sharper and presented lesser background compared to the isotopic labeling method (Fig. 6A). Unlike isotopic probes, digoxigenin-labeled probes are innocuous, can be easily disposed of, can be stored for long periods, and can even be reused. It is worth noting, however, that this nonisotopic detection method requires some minor precautions. First, the nylon membrane used for this type of detection must bear a specific density and homogeneous distribution of positive charge. Among membranes we tested, the one sold by Roche Molecular Biochemicals, unquestionably gave the best results. Secondly, care should be taken with the manipulation


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of the membrane. The use of tweezers is strongly recommended in order to reduce nonspecific spots and background. As seen in Fig. 6B, in spite of taking every precaution, some small spots are still observed on the “chemiluminogram.” These might be explained by the powder from gloves. An alternative explanation for these spots could be the presence of nondissolved crystals in the antibody solution (to minimize this problem, this solution can be spun for 15–30 s before use) or in the detection buffer. However, the use of an appropriate membrane and meticulous manipulations can produce very good results with the nonisotopic detection method. 12. To avoid long probes, (i.e., greater than 200 bp), plasmid DNA is cut with an appropriate restriction enzyme (e.g., see ref. 16). If a third primer (primer 3) is used to make the probe, it should be selected from the same strand as the amplification primer (primer 2) just 5' to primer 2 sequence and with no more than seven to eight bases of overlap on this primer, and should have a Tm of 60–68°C. As first reported by Hornstra and Yang (41,51,52), we simply use the primer 2 employed in the amplification step and we produce the probe from PCR products. Such probes cost less (no primer 3), are more convenient (the preparation of the PCR products permits the testing of primers) and simplify the assay because no cloning requirement is needed as long as the sequence is known. 13. The bottom of a capless 0.625-mL siliconized microtube can be easily pierced with a heated needle. It is important to emphasize that the hole should be made as small as possible for the column to efficiently retain agarose. The pierced microtube is packed with wetted glass wool. Three successive centrifugation steps of 1 min each at 16,000g are necessary to compact and dry the glass wool. The water is recuperated in a capless 1.5-mL microtube. If glass wool is found with the effluent, the column should be discarded. A final 5-min centrifugation at 16,000g should be carried out to ensure the glass wool is fully compacted and dry. The glass wool column is stored at room temperature in a new capless 1.5-mL microtube and covered with a plastic film to protect the column from dust. In this way, the column can be stored indefinitely until it is used. 14. To verify whether digoxigenin was incorporated in the probe, use an aliquot of 1 µL from the 100-µL probe preparation and pipet onto a small piece of positively charged membrane (see Subheading 2.5.4., item 13). Expose the membrane to 1000 J/m2 of 254-nm UV to crosslink the probe onto the membrane. Place the membrane in a Petri dish and add 15 mL of buffer 1 (see Subheading 2.5.5.2., item 5). Discard the buffer 1, add 20 mL of buffer 2 (see Subheading 2.5.5.2., item 6), and place the dish on a shaker for 10 min at room temperature. Discard the buffer 2, add 20 mL of digoxigenin-antibody coupled with a peroxidase (antiDigoxigenin-AP, Roche Molecular Biochemicals, cat. no. 1 093 274) diluted 1:5 000 in buffer 2. Incubate 10 min at room temperature. Add 20 mL of buffer 1 in a new Petri dish, transfer the membrane to this new dish and wash the membrane for 10 min at room temperature. Always manipulate the membrane with tweezers (see Note 11). Remove the buffer 1 and add 20 mL of buffer 3 (see Subheading 2.5.5.2., item 9). Wait 5 min to allow the membrane to reach the appropriate pH

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Drouin et al. (pH 9.5) for the detection. During this time, prepare the detection solution by adding 90 µL of NBT (4-Nitroblue tetrazolium chloride, Roche Molecular Biochemicals, cat. no. 1 383 213) and 70 µL of BCIP (X-phosphate/5-bromo-4chloro-3-indolyl-phosphate, Roche Molecular Biochemicals, cat. no. 1 383 221) to 20 mL of buffer 3. Discard buffer 3 and add the detection solution to the dish containing the membrane and place it in a dark room. Check occasionally and monitor the appearance of staining. If no staining appears after 1 h, this means that the incorporation of DIG was not efficient. The detection solution is very toxic, manipulate it carefully and eliminate this solution as toxic waste.

Acknowledgments The authors wish to thank Dr. Elliot A. Drobetsky for his precious help in editing this text and for exciting LMPCR discussions. We are grateful to Mrs. Nancy Dallaire, Isabelle Paradis, and Nathalie Bastien for their technical assistance and valuable contribution to the development of the LMPCR technology. This work was supported by the Medical Research Council of Canada (MRC) and the Canadian Genetic Diseases Network (MRC/NSERC NCE program). R. Drouin is presently a research scholar (“Chercheur-boursier”) of the “Fonds de la Recherche en Santé du Québec” (FRSQ). References 1. Pfeifer, G. P., Tanguay, R. L., Steigerwald, S. D., and Riggs, A. D. (1990) In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK–1. Genes Dev. 4, 1277–1287. 2. Pfeifer, G. P. and Riggs, A. D. (1991) Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permealized cells using DNase I and ligation-mediated PCR. Genes Dev. 5, 1102–1113. 3. Chen, C.-J., Li, L. J., Maruya, A., and Shively, J. E. (1995) In vitro and in vivo footprint analysis of the promoter of carcinoembryonic antigen in colon carcinoma cells: effects of interferon γ treatment. Cancer Res. 55, 3873–3882. 4. Tornaletti, S. and Pfeifer, G. P. (1995) UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes. J. Mol. Biol. 249, 714–728. 5. Mueller P. R. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786. 6. Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813. 7. Pfeifer, G. P., Steigerwald, S. D., Hansen, R. S., Gartler, S. M., and Riggs, A. D. (1990) Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability. Proc. Natl. Acad. Sci. USA 87, 8252–8256.


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8. Pfeifer, G. P., Drouin, R., Riggs, A. D., and Holmquist, G. P. (1992) Binding of transcription factors creates hot spots for UV photoproducts in vivo. Mol. Cell. Biol. 12, 1798–1804. 9. Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995. 10. Pfeifer, G. P. (1992) Analysis of chromatin structure by ligation-mediated PCR. PCR Methods Appl. 2, 107–111. 11. Pfeifer, G. P. and Riggs, A. D. (1993) Genomic footprinting by ligation mediated polymerase chain reaction, in PCR Protocols: Current Methods and Applications (White, B., ed.), Humana, Totowa, NJ, pp. 153–168. 12. Pfeifer, G. P. and Riggs, A. D. (1993) Genomic sequencing, in DNA Sequencing Protocols (Griffin, H. G. and Griffin, A. M., eds.), Humana, Totowa, NJ, pp. 169–181. 13. Pfeifer, G. P., Singer-Sam, J., and Riggs, A. D. (1993) Analysis of methylation and chromatin structure. Methods Enzymol. 225, 567–583. 14. Gao, S., Drouin, R., and Holmquist, G. P. (1994) DNA repair rates mapped along the human PGK1 gene at nucleotide resolution. Science 263, 1438–1440. 15. Tornaletti, S. and Pfeifer, G. P. (1994) Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science 263, 1436–1438. 16. Rodriguez, H., Drouin, R., Holmquist, G. P., O’Connor, T. R., Boiteux, S., Laval, J., Doroshow, J. H., and Akman, S. A. (1995) Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J. Biol. Chem. 270, 17,633–17,640. 17. Drouin, R. and Therrien, J.-P. (1997) UVB-induced cyclobutane pyrimidine dimer frequency correlates with skin cancer mutational hotspots in p53. Photochem. Photobiol. 66, 719–726. 18. Rozek, D. and Pfeifer, G. P. (1993) In vivo protein–DNA interactions at the c-jun promoter: preformed complexes mediate the UV response. Mol. Cell. Biol. 13, 5490–5499. 19. Cartwright, I. L. and Kelly, S. E. (1991) Probing the nature of chromosomal DNA– protein contacts by in vivo footprinting. BioTechniques 11, 188–203. 20. Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Methods Enzymol. 65, 499–560. 21. Chin P. L., Momand, J., and Pfeifer, G. P. (1997) In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation. Oncogene 15, 87–99. 22. Angers, M., Drouin, R., Bachvarova, M., Paradis, I., Marceau, F., and Bachvarov, D. R. (2000) In vivo protein–DNA interactions at the kinin B1 receptor promoter: no modification upon interleukin-1 beta or lipopolysaccharide induction. J. Cell. Biochem. 78, 278–296. 23. Becker, M. M. and Wang, J. C. (1984) Use of light for footprinting DNA in vivo. Nature 309, 682–687. 24. Pfeifer, G. P. and Tornaletti, S. (1997) Footprinting with UV irradiation and LMPCR. Methods 11, 189–196.

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25. Pfeifer, G. P., Chen, H. H., Komura, J., and Riggs, A.D. (1999) Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction. Methods Enzymol. 304, 548–571. 26. Cadet, J., Anselmino, C., Douki, T., and Voituriez, L. (1992) Photochemistry of nucleic acids in cells. J. Photochem. Photobiol. B: Biol. 15, 277–298. 27. Mitchell, D. L. and Nairn, R. S. (1989) The biology of the (6–4) photoproducts. Photochem. Photobiol. 49, 805–819. 28. Holmquist, G. P. and Gao, S. (1997) Somatic mutation theory, DNA repair rates, and the molecular epidemiology of p53 mutations. Mutat. Res. 386, 69–101. 29. Pfeifer, G. P., Drouin, R., Riggs, A. D., and Holmquist, G. P. (1991) In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6–4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction. Proc. Natl. Acad. Sci. USA 88,1374–1378. 30. Gale, J. M. and Smerdon, M. J. (1990) UV induced (6–4) photoproducts are distributed differently than cyclobutane dimers in nucleosomes. Photochem. Photobiol. 51, 411–417. 31. Gale, J. M., Nissen, K. A., and Smerdon, M. J. (1987) UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases. Proc. Natl. Acad. Sci. USA 84, 6644–6648. 32. Mitchell, D. L., Nguyen, T. D., and Cleaver, J. E. (1990) Nonrandom induction of pyrimidine–pyrimidone (6–4) photoproducts in ultraviolet-irradiated human chromatin. J. Biol. Chem. 265, 5353–5356. 33. Rigaud, G., Roux, J., Pictet, R., and Grange, T. (1991) In vivo footprinting of rat TAT gene: dynamic interplay between the glucocorticoid receptor and a liverspecific factor. Cell 67, 977–986. 34. Miller, M. R., Castellot, J. J., and Pardee, A. B. (1978) A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry 17, 1073–1080. 35. Contreras, R. and Fiers, W. (1981) Initiation of transcription by RNA polymerase II in permeable SV40-infected CV-1 cells; evidence of multiple promoters for SV40 late transcription. Nucleic Acids Res. 9, 215–236. 36. Tanguay, R. L., Pfeifer, G. P., and Riggs, A. D. (1990) PCR-aided DNase I footprinting of single-copy gene sequences in permeabilized cells. Nucleic Acids Res. 18, 5902. 37. Törmänen, V., Pfeifer, G. P., Swiderski, P. M., et al. (1992) Extension product capture improves genomic sequencing and DNase I footprinting by legation-mediated PCR. Nucleic Acids Res. 20, 5487–5488. 38. Tornaletti, S., Bates, S., and Pfeifer, G. P. (1996) A high-resolution analysis of chromatin structure along p53 sequences. Mol. Carcinogen. 17, 192–201. 39. Szabo, P. E., Pfeifer, G. P., and Mann, J. R. (1998) Characterization of novel parent-specific epigenetic modifications upstream of the imprinted mouse H19 gene. Mol. Cell. Biol. 18, 6767–6776. 40. Garrity, P. A. and Wold, B. J. (1992) Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proc. Natl. Acad. Sci. USA 89, 1021–1025.


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41. Hornstra, I. K. and Yang, T. P. (1994) High resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors. Mol. Cell. Biol. 14, 1419–1430. 42. Angers, M., Cloutier, J.-F., and Drouin, R. (2000) The effectiveness of Pfu exo– DNA polymerase in ligation-mediated PCR is mainly modulated by the ratio of DNA molecules per unit of enzyme. Submitted. 43. Drouin, R., Gao, S., and Holmquist, G. P. (1996) Agarose gel electrophoresis for DNA damage analysis, in Technologies for Detection of DNA Damage and Mutations (Pfeifer, G. P., ed.), Plenum, New York, pp. 37–43. 44. Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 17, 8543–8551. 45. Drouin, R., Rodriguez, H., Holmquist, G. P., and Akman, S. A. (1996) Ligationmediated PCR for analysis of oxidative DNA damage, in Technologies for Detection of DNA Damage and Mutations (Pfeifer, G. P., ed.), Plenum, New York, pp. 211–225. 46. Mueller PR, Wold, B. (1991) Ligation-mediated PCR: applications to genomic footprinting. Methods 2, 20–31. 47. Iverson, B. L. and Dervan, P. B. (1987) Adenine specific DNA chemical sequencing reaction. Nucleic Acids Res. 19, 7823–7830. 48. Zhang, L. and Gralla, J. D. (1989) In situ nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site. Genes Dev. 3, 1814–1822. 49. Rychlik, W. (1993) Selection of primers for polymerase chain reaction, in PCR Protocols: Current Methods and Applications (White, B., ed.), Humana, Totowa, NJ, pp. 31–40. 50. Fry, M. and Loeb, L. A. (1994) The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure. Proc. Natl. Acad. Sci. USA 91, 4950–4954. 51. Hornstra, I. K. and Yang, T. P. (1992) Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation. Mol. Cell. Biol. 12, 5345–5354. 52. Hornstra, I. K. and Yang, T. (1993) In vivo footprinting and genomic sequencing by ligation-mediated PCR. Anal. Biochem. 213, 179–193.

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14 Identification of Protein–DNA Contacts with Dimethyl Sulfate Methylation Protection and Methylation Interference Peter E. Shaw and A. Francis Stewart 1. Introduction Dimethyl sulfate (DMS) is an effective and widely used probe for sequencespecific protein–DNA interactions. It is the only probe routinely used both for in vitro (methylation protection, methylation interference) and in vivo (DMS genomic footprinting) applications because it rapidly reacts with DNA at room temperature and readily penetrates intact cells (1). DMS methylates predominantly the 7-nitrogen of guanine and the 3-nitrogen of adenine. Thus reactivity with G residues occurs in the major groove and with A residues in the minor groove. In standard Maxam and Gilbert protocols (2), the methylated bases are subsequently converted to strand breaks and displayed on sequencing gels. Methylation protection and interference are essentially combinations of the gel retardation assay or electrophoretic mobility shift assay (EMSA) (3,4) (Chapter 2) with the DMS reaction of the Maxam and Gilbert sequencing procedure. Protein–DNA interactions are reflected either as changes in DMS reactivities caused by bound protein (methylation protection) or as selective protein binding dictated by methylation (methylation interference). In methylation protection, protein is first bound to DNA that is uniquely end labeled and the complex is reacted with DMS. DMS reactivities of specific residues are altered by bound protein either by exclusion, resulting in reduced methylation, or by increased local hydrophobicity, resulting in enhanced methylation, or by local DNA conformational changes, such as unwinding, resulting in altered reactivity profiles (5–7). After the DMS reaction, free DNA is separated from protein-bound DNA by gel retardation and both DNA From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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fractions are recovered from the gel. Methylated residues are converted into strand scissions and the free and bound DNA fractions are compared on a sequencing gel. A complete analysis requires the examination of both strands. This is accomplished by preparing two DNA probes, each uniquely labeled at one end, and carrying both probes through the protocols. A binding site characterized by methylation protection will therefore appear as a cluster of altered DMS reactivities. In methylation interference (8,9), DNA is first reacted with DMS, purified and then presented to protein. Under the reaction conditions used methylation is partial, yielding approximately one methylated base per DNA molecule. Thus, the protein is presented with a mixture of DNA molecules that differ with respect to the positions of methyl groups. Some methyl groups will interfere with protein binding because they lie in or near the binding site. Gel retardation separates the mixture into two fractions: free DNA, which, as long as DNA is in excess over binding activity, represents the total profile of methylation reactivity, and bound DNA, which will not contain any molecules with methyl groups incompatible with binding. Both free and bound DNA fractions are recovered, methylated residues are converted to strand scissions, and the fractions are compared on a sequencing gel. The binding site is observed as the absence of bands in the bound sample corresponding to the positions where methylation interferes with binding. It is obvious that these two uses of DMS may not deliver identical results. For example, Fig. 1 presents a comparison obtained from experiments with the serum response element binding factors p67SRF p62TCF and their binding site in the human c-fos promoter (SRE). Because the use of DMS in vivo for genomic footprinting is limited to the equivalent of methylation protection, a direct comparison between in vivo and in vitro data excludes the more widely used methylation interference assay. The two techniques are, however, very similar in practical terms and thus are presented together. Both techniques rely on preestablished conditions that permit a protein–DNA complex to be resolved in a gel retardation assay (Chapter 2) and on chemical DNA sequencing methodologies, for which the reader is advised to consult ref. 2 for a detailed treatment. 2. Materials 1. Dimethyl sulfate (DMS) (Merck), analytical grade. 2. Piperidine (Sigma), analytical grade; use freshly made 1:10 dilution in doubledistilled water. 3. Phenol/chloroform 50% v/v, buffered with 50 mM Tris-HCl, pH 8.0. 4. NA45 paper (Schleicher & Schuell). 5. 3MM paper (Whatman) or GB 002 paper (Schleicher & Schuell).


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Fig. 1. Comparison of methylation interference and protection patterns formed by factors binding at the c-fos serum response element (SRE) in vitro and in vivo. G residues identified by methylation interference (9), methylation protection and in vivo genomic footprinting (7) are indicated. An additional G on both the upper and lower strands is implicated in the protein–DNA interaction by methylation protection. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17.

Electrophoresis equipment suitable for gel retardation or EMSA. Electroblotting apparatus for Western transfer (e.g., Bio-Rad Trans-Blot). Standard DNA sequencing gel electrophoresis equipment. Vacuum gel drier (optional). TBE buffer: 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA. Make 10X stock as 108 g Tris base, 55 g boric acid, and 40 mL of 0.5 M EDTA pH 8.3 per liter. NA45 elution buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 M NaCl. Carrier DNA: Salmon testis DNA or calf thymus DNA (Sigma), dissolved at 3 mg/mL in 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, and sheared. Sequencing loading buffer: 90% formamide, 10 mM EDTA, and 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol blue. Gel retardation loading buffer: 20% Ficoll, 20 mM EDTA, 0.1% w/v bromophenol blue. 2X DMS buffer: 120 mM NaCl, 20 mM Tris-HCl, pH 8.0, 20 mM MgCl2, and 2 mM EDTA. DMS stop buffer: 1.5 M NaAc, pH 7.0, and 1 M 2-mercapto-ethanol, store frozen. X-ray film (e.g., Kodak X-Omat) or imaging plate for phosphorimager.

3. METHOD 3.1. Methylation Protection 1. Incubate 300,000 cpm of uniquely end-labeled DNA probe (see Note 1) and a corresponding amount of protein together in a total volume of 100 µL, as previously optimized for gel retardation analysis. 2. Add 1 µL of DMS and incubate at room temperature (the incubation time depends on the length of the DNA probe and is empirical; as a guide for a 200-bp fragment, 1.5 min, for a 50-bp oligonucleotide duplex, 3 min). 3. Add 1/10 vol of 250 mM dithiothreitol (DTT), mix gently, add 1/10 vol of gel retardation loading buffer, mix gently, load onto a 2-mm-thick retardation gel in

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5.

6.

7.

8.

9a.

9b.

Shaw and Stewart 1X TBE (or an alternative buffer as determined to be best for the given complex) and run as optimized for analytical gels. The load may need to be spread over up to 10 times more well area as compared with an analytical retardation assay (see Note 2). After electrophoresis, separate the glass plates carefully so that the gel adheres to one plate and cover the gel with cling film. Expose to X-ray film long enough to reveal complexes clearly (i.e., 6 h to overnight). The alignment of the film to the gel must be reliably marked. Put the developed film on a light box. Remove the gel from the cling film and realign it on the X-ray film. Cut pieces of NA45 paper sufficiently large to cover individual complexes in the gel yet small enough to fit into 1.5-mL tubes when rolled up. Wet the paper pieces in retardation gel running buffer and, with the help of tweezers, position one over each complex of interest in the gel, as visualized from the underlying film. Also position a similar sized piece of paper over (some of) the uncomplexed DNA. NA45 paper can be labeled with pencil before wetting. Carefully cover the gel and paper pieces with two sheets of 3MM paper wetted in 1X TBE (or alternative gel running buffer from step 3). Lay a ScotchBrite pad from the electroblotting apparatus on top of the paper and turn the gel over. Carefully remove the second glass plate, cover the other side of the gel with 3MM paper and ScotchBrite as before and insert the package into an electrotransfer apparatus as described in the manufacturer’s instructions with the NA45 paper toward the anode. Transfer in 1X TBE (or the alternative retardation gel buffer) at 80 V for 1.5 h (see Note 3). Stop the transfer, unpack the gel carefully with the NA45 paper on top and transfer each piece to a labeled 1.5-mL tube containing 600 µL of elution buffer (check that the radioactivity has transferred to the paper). Incubate at 70°C for 1 h. Remove NA45 paper from each tube, check that at least half the radioactivity has eluted into the buffer (do not expect quantitative elution, but at least 50% should come off), add 20 µg carrier DNA, extract with phenol/chloroform and precipitate with 1 volume of isopropanol. Wash precipitate once in 70% ethanol and dry under vacuum. (See Notes 4 and 5.) To reveal modified Gs: Dilute piperidine 1:10 in water and add 50 µL to each pellet. Vortex briefly and incubate at 90o C for 30 min (tubes must be clamped or weighted down to prevent the lids opening); then dry under a good vacuum. Take up the samples in 100 µL of water and repeat the drying process. This strand scission protocol should not convert methylated A residues into strand breaks. It is often observed, however, that breakages at As do occur with reasonable efficiency. To reveal modified As and Gs: The following modification will produce efficient cleavage at both methyl-G and methyl-A residues. After the preparative retardation gel, resuspend the dried, purified DNA in 30 µL of 10 mM sodium phosphate pH 6.8, and 1 mM EDTA. Incubate for 15 min at 92°C. Then add 3 µL of 1 M NaOH and incubate for 30 min at 92°C, followed by 320 µL of 500 mM NaCl,


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50 µg/mL carrier DNA, and 900 µL ethanol. Chill and centrifuge to pellet the radioactivity. Wash the pellet in 70% ethanol and dry. 10. Measure the Cerenkov counts in each tube, then redissolve the samples in water (e.g., 10 cpm/µL) and transfer equivalent counts (1000 cpm in each case would be optimal) from each into fresh tubes. Dry down and redissolve in 5 µL sequencing loading buffer. 11. Prepare and pre-electrophorese a standard sequencing gel (5–12% acrylamide, depending on probe length). Denature probes at 95oC for 5 min, snap cool in ice and load onto the gel. Run the gel until optimal separation of sequence is achieved. (See Notes 6–8.) 12. Stop electrophoresis, remove the gel from the tank and lift off one glass plate. Fix the gel in 20% ethanol and 10% acetic acid for 10 min. Drain briefly and then overlay the gel with two sheets of 3MM paper and carefully peel it off the glass plate. Cover the gel surface with cling film and dry on a vacuum gel drier (see Note 9). Expose the dry gel to X-ray film with intensifying screens as necessary, or to an imaging plate.

3.2. Methylation Interference 1. Mix 300,000 cpm of end-labeled probe (see Note 1); 100 µL of 2X DMS buffer and water to 200 µL. Add 2 µL of DMS and incubate at room temperature (the same guidelines as given in Subheading 3.1.2. apply for the reaction time). Stop the reaction by the addition of 50 µL cold DMS stop mix and precipitate with 850 µL cold ethanol. Redissolve in 200 µL cold 0.3 M NaAc pH 7.0, add 700 µL cold ethanol, and reprecipitate. Wash twice in 80% ethanol and redissolve the probe in water or binding buffer (about 20,000 cpm/µL). 2. Incubate the probe with protein for gel retardation as previously optimized for gel retardation analyses of the complexes in question in a total volume of 100 µL. 3. Add 1/10 vol of gel retardation loading buffer, mix gently and load onto a 2-mm-thick retardation gel in 1X TBE (or alternative buffer); then, run as optimized for analytical gels. However, the load should be spread over up to 10 times more well area (see Note 2). 4. Continue with step 4 and all subsequent steps as described for methylation protection (Subheading 3.1.).

4. Notes 1. To have sufficient counts to complete the procedure, proceed with at least 10 times the amount of material required for a simple gel retardation analysis (i.e., at least 300,000 cpm). 2. A common difficulty with these methods is the persistence of contaminants that accompany DNA after the preparative retardation gel. These contaminants interfere with the migration of DNA on the sequencing gel, producing blurred and distorted patterns. In order to minimize this problem it is worth ascertaining the load limit of the retardation gel so that the protein–DNA complex will not smear but be well resolved and therefore concentrated in the gel before elution.

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3. It is also possible to use a semidry electrotransfer apparatus (e.g., Bio-Rad Transblot SD) to transfer the DNA from the gel retardation gel onto NA45 paper. In this case, both the transfer time and potential are reduced. 4. In some instances, it may prove difficult to elute the DNA from the NA45 paper, in which case raising the salt concentration or the temperature may improve elution. (Extending the incubation time does not seem to help.) If not, the batch of NA45 may be to blame or it is even conceivable that the DNA–protein complex in question is adsorbed too tightly onto the paper. It is not possible to phenol extract the NA45 paper in order to remove bound protein–DNA. 5. Retain the isopropanol supernatants until you are sure the samples have precipitated quantitatively. Add more carrier DNA if required. 6. It is similarly advisable to load as little material onto the sequencing gel as practicable. With the advent of the phosphorimager, the lower limit for the sequencing gel is well under 1000 cpm/lane. 7. If the end-labeled DNA fragment is relatively long and multiple binding sites are to be resolved, a gradient or wedge sequencing gel can be used in step 11 of Subheading 3.1. 8. An appropriate complement for the final result is to perform the Maxam and Gilbert G+A reactions on the end-labeled probe. On the sequencing gel, these reactions should provide unambiguous sequence information and, in case difficulties are encountered, clues as to the steps that are problematic. 9. It is not essential to dry down the sequencing gel because after one glass plate has been removed, it can be covered with cling film and exposed to X-ray film at –70°C with one screen. This alternative should only be considered if the signal is sufficiently strong or if a gel drier is not available.

References 1. Church, G. M. and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995. 2. Maxam, A. and Gilbert, W. (1980) Sequencing end-labelled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499–560. 3. Fried, A. and Crothers, D. M. (1981) Equilibria and kinetics of lac repressor– operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525. 4. Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of protein to specific DNA regions: application to components of the E. coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3059. 5. Gilbert, W., Maxam, A., and Mirzabekov, A. (1976) Contacts between the LAC repressor and DNA revealed by methylation. in Control of Ribosome Biosynthesis, Alfred Benzon Symposium IX (Kjelgaard, N. O. and Maaloe, O., eds.), Academic, New York, pp. 139–148. 6. Johnsrud, L. (1978) Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc. Natl. Acad. Sci. USA 75, 5314–5318.


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7. Herrera, R. E., Shaw, P. E., and Nordheim, A. (1989). Occupation of the c-fos serum response element in vivo by a multi-protein complex is unaltered by growth factor induction. Nature 340, 68–70. 8. Siebenlist, U. and Gilbert, W. (1980) Contacts between E. coli RNA polymerase and an early promoter of phage T7. Proc. Natl. Acad. Sci. USA 77, 122–126. 9. Shaw, P. E., Schröter, H., and Nordheim, A. (1989). The ability of a ternary complex to form over the serum response element correlates with serum inducibility of the human c-fos promoter. Cell 56, 563–572.

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15 Ethylation Interference Iain W. Manfield and Peter G. Stockley 1. Introduction Structural studies of DNA–protein complexes have now made it clear that specific sequence recognition in these systems is accomplished in two ways, either directly by the formation of hydrogen bonds to base-pair edges from amino acid side chains located on a DNA-binding motif, such as a helix–turn– helix, or indirectly as a result of sequence-dependent distortions of the DNA conformation (1). These contacts occur in the context of oriented complexes between macromolecules that juxtapose the specific recognition elements. As part of these processes, proteins make a large number of contacts to the phosphodiester backbone of DNA, as was predicted from biochemical assays of the ionic strength dependence of DNA binding. Contacts to phosphate groups can be inferred by the ethylation interference technique (2). Ethylnitrosourea (EtNU) reacts with DNA to form, principally, phosphotriester groups at the nonesterified oxygens of the otherwise phosphodiester backbone. Minor products are the result of the reactions of EtNU with oxygen atoms in the nucleotide bases themselves (see Note 1). Under alkaline conditions and at high temperature, the backbone can be cleaved at the site of the modification to form a population of molecules carrying either 3'-OH or 3'-ethylphosphate groups. The length of the ethyl group (approx 4.5 Å) means that at a number of positions along a DNA molecule encompassing the binding site for a protein, complex formation will be inhibited by the presence of such a modification. At other sites, outside the binding site, no interference with protein binding at the specific site will be observed. Addition of the DNA-binding protein to a randomly ethylated DNA sample, followed by some procedure to separate the complexes formed from unbound DNA, will fractionate the DNA sample into From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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those molecules able to bind protein with high affinity and those for which the ethylation has lowered the affinity (Fig. 1). In practice, modification at different sites produces molecules with a spectrum of affinities for the protein. It is, therefore, not possible to prove conclusively that a particular phosphate is contacted by the protein, but only that ethylation at that site interferes with complex formation. Only occasionally are large amounts of pure protein readily available for in vitro biochemical assay of DNA-binding activity and, often, only small amounts of crude nuclear extracts are available. In many commonly used assays, complex formation could not easily be detected in such situations. For example, using DNase I or hydroxyl radicals, a high level of binding-site occupancy is required for a footprint to be detected. Fractional occupancy is readily detected by gel retardation of complexes (electrophoretic mobility shift [EMSA]; see Chapter 2) but offers only limited characterization of the details of the protein–DNA interaction. Interference techniques, such as the ethylation and hydroxyl radical interference (see Chapter 16) techniques (3), do allow the molecular details of complex formation to be studied even when only small amounts of crude protein are available (4). Whatever the level of saturation, DNA fragments modified at sites reducing the affinity of protein for DNA are less likely to form complexes. Therefore, the bound fraction on gel retardation assays will always give an indication of the sites that do not inhibit complex formation when modified. The groups on the DNA recognized by the protein can then be inferred. Ethylation can also be used to analyze RNA–protein complexes (5,6). We have used the ethylation interference technique to probe the interaction of the Escherichia coli methionine repressor, MetJ, with its binding site in vitro. Binding sites for MetJ consist of two, or more, immediately adjacent copies of an 8-bp site with the consensus sequence 5'-AGACGTCT-3', which has been termed a “met box.” X-ray crystallography has been used to determine the structure of the MetJ dimer, the complex with corepressor, S-adenosyl methionine (SAM), and a complex of the holorepressor with a 19-mer oligonucleotide containing two met boxes (7,8). The structure of the protein–DNA complex in the crystal reveals two MetJ dimers (one per met box) binding to the DNA by insertion of a β-ribbon into the major groove. The general features of this model are corroborated by the results of the ethylation interference experiments and by data from a range of other footprinting techniques. 2. Materials 2.1. Preparation of Radioactively End-Labeled DNA 1. Plasmid DNA carrying the binding site for a DNA-binding protein on a convenient restriction fragment (usually 5%). It is necessary to account for this when calculating the ANS concentration at each point in the titration. 6. See Chapter 33 for a more detailed discussion of the inner filter effect. As a guide, for excitation at 370 nm in an aqueous buffer, 52 µM ANS has an OD370 of 0.11 in a 0.4-cm path-length cuvet. This gives rise to an inner filter correction of 1.14 using Eq. 1. 7. The binding curve generated for ANS can, in principle, take many forms. The shape will depend on the number and relative affinity of ANS binding sites on the protein. If the protein contains high-affinity sites, the curve may be biphasic and may allow the stoichiometry of the strong interaction to be determined. A more likely situation is that there will be numerous ANS binding sites with differing but weak affinities (Kd > 100 µM). The result of this is a curved plot similar to Fig. 3. 8. The concentration of the ANS solution used in the titration must be determined empirically from the previous experiments. It should be high enough to ensure that a good fraction of the ANS binding sites on the protein are occupied (as determined in Subheading 3.1.), 9. As well as direct excitation of the fluorescent probe (i.e., with an excitation wavelength of 370 nm for ANS), it may be possible to investigate energy transfer

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effects between aromatic amino acid residues in the protein and the bound probe. If the excitation is performed at 280 nm to excite both tyrosine and tryptophan, energy transfer to ANS will be apparent from the emission spectrum between 400 and 600 nm. In principle this effect could also be useful to for following displacement of ANS in a titration with DNA. 10. A further extension of the fluorescent probe approach is to employ the covalent probe 5-((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) (5). This reagent reacts with accessible cysteine residues in the protein and has a higher quantum yield than ANS in aqueous solution. One can look at the emission spectrum of the bound probe, or it may be possible to observe energy transfer from aromatic residues in the protein. Any of these fluorescence characteristics could change when the DNA is bound if the probe is located near the DNA-binding site. We have used this technique to study the interaction of M.EcoR124I with DNA and found that energy transfer from the protein to the bound probe decreased by over 30% when DNA was bound. As long as the presence of the probe does not inhibit binding, then titrations with DNA can be used to produce DNA-binding curves. However if the probe does inhibit, this can also be informative; the labelled cysteine(s) can be identified by peptide mapping by analogy with the methods reported in Chapter 20.

References 1. Brand, L. and Gohlke, J. R. (1972) Fluorescence probes for structure. Annu. Rev. Biochem. 41, 843–868. 2. Secnik, J., Wang, Q., Chang, C. M., and Jentoft, J. E. (1990) Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'disulfonic acid. Biochemistry 29, 7991–7997. 3. York, S. S., Lawson, R. C., Jr., and Worah, D. M. (1978) Binding of recrystallized and chromatographically purified 8-anilino-1-naphthalenesulfonate to Escherichia coli lac repressor. Biochemistry 17, 4480–4486. 4. Taylor, I., Patel, J., Firman, K., and Kneale, G. (1992) Purification and biochemical characterization of the EcoR124 type I modification methylase. Nucleic Acids Res. 20, 179–186. 5. Kelsey, D. E., Rounds, T. C., and York, S. S. (1979) lac repressor changes conformation upon binding to poly[dA-T)]. Proc. Natl. Acad. Sci. USA 76, 2649–2653.

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19 Site-Directed Cleavage of DNA by Linker Histone-Fe(II) EDTA Conjugates David R. Chafin and Jeffrey J. Hayes 1. Introduction The ordered and regular packaging of eukaryotic DNA within the chromatin complex allows the efficient utilization of this substrate for nuclear processes such as DNA replication, transcription, recombination, and repair (1,2). Thus, an understanding of the organization of protein–DNA interactions and associations within the chromatin complex is a prerequisite for a complete molecular description of these processes. For example, the linker histone protein plays crucial roles in the stability and organization of the chromatin fiber (3,4). This multidomain protein undoubtedly makes complicated and diverse but poorly understood interactions within the chromatin fiber (1). Currently, there is disagreement regarding the site of association of the globular domain of this protein within the nucleosome proper (5,6). Moreover, the molecular interactions and chemical activities of its N- and C-terminal tails are most likely modulated by the multiple posttranslational phosphorylation events known to occur within these domains (1,2). Thus, the linker histone tails represent critical points for signal transduction within the chromatin complex likely to be manifested as structural alterations within chromatin. To elucidate the multiple interactions between the linker histone protein and several model chromatin complexes, we have opted for a site-directed chemical cleavage methodology originally introduced by Ebright and colleagues and Fox and colleagues (7,8). This method relies on targeting a DNA cleavage reagent via the unique nucleophilic properties of a cysteine sulfhydryl engineered at rationally selected locations within the protein of interest. The linker histone protein is a particularly suitable candidate for this type of approach because only in one rare instance (9) has this protein been found to contain a From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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cysteine residue. The single sulfhydryl group within the protein is modified with a bifunctional reagent that contains a cysteine-specific moiety at one end and an iron(II)-based DNA cleavage reagent at the other (Fig. 1) (7,8,10–12). The protein is then assembled into the chromatin complex of interest and DNA cleavage is initiated by standard Fenton chemistry (7,8). The DNA from such complexes is prepared and the location of DNA cleavage is mapped to singlebase-pair resolution on DNA sequencing gels. 2. Materials 2.1. Construction of Cysteine Substituted Protein

2.1.1. Point Mutation by PCR 1. Oligonucleotide primers: Two oligonucleotide primers complimentary to the 5' and 3' ends of the sequence to be amplified are needed. In addition, if the codon to be altered is located more than approx 10–15 nucleotides from the end of the coding sequence, one additional primer is needed that must contain the sequence substitutions for the altered codon flanked by 12–15 nucleotides of complementary sequence on each side. Store at –20°C. 2. 10X stock containing all four dNTPs at 10 mM concentration each. 3. A source of clean reliable 18 meg Ω water, free of chemical contaminants. 4. 10X polymerase chain reaction (PCR) buffer; can be obtained commercially from the supplier of the PCR enzyme of choice. 5. Vent or Taq DNA polymerase; can be obtained from commercial sources.

2.1.2. Ligation and Transformation of PCR Insert into DH5α or BL21 Cells 1. DH5α or BL21 cells can be obtained commercially or prepared in competent form; store at –70°C. 2. Luria–Bertani (LB) medium, sterile. 3. 1000X stock of ampicillin (100 mg/mL). 4. LB–agar plates containing 0.1 mg/mL ampicillin.

2.1.3. Overexpression and Purification of Mutant Histone Proteins 1. 2. 3. 4. 5. 6. 7. 8.

100X (0.2 M) stock of isopropyl β-D-thiogalactopyranoside (IPTG). Luria–Bertani (LB) medium, sterile. 10 mg/mL lysozyme solution. 1000X PMSF (phenylmethylsulfonyl fluoride): 100 mM in ethanol. Triton-X100 detergent. A 2-M solution of NaCl. A 50% (v/v) slurry of Bio-Rex 70, 100-mesh chromatography resin (Bio-Rad). 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA solutions containing 0.5 M, 0.6 M, 1.0 M, and 2.0 M NaCl.


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Fig. 1. Lysine at position 59 within the globular domain of histone H1 was changed to a cysteine residue (K59C, top). The free sulfhydryl group on the cysteine residue was coupled to the DNA cleavage reagent EPD (middle). Hydroxyl radicals were produced from the Fe2+ center contained within the EPD moiety by standard Fenton chemistry (bottom and equation).

2.2. Reduction and Modification of Cysteine-Substituted Proteins with EDTA-2-aminoethyl 2-pyridyl disulfide (EPD) 1. 1 M stock of DTT (dithiothreitol), made fresh; store at –20°C. 2. A 50% slurry of Bio-Rex 70, 100–200 mesh chromatography resin (Bio-Rad). 3. 10 mM Tris-HCl, pH 8.0 solutions containing 0.5 M, 0.6 M, 1.0 M, and 2.0 M NaCl.

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4. 0.3 M stock of EPD synthesized according to refs. 7 and 8. Alternatively, iodoacetamido-1,10 phenanthroline–Cu2+ can be employed in place of EPD (Molecular Probes, Eugene, OR). 5. Disposable 10 mL plastic chromatography columns (Bio-Rad). 6. Coomassie blue stain: 45% methanol, 10% acetic acid, and 0.25% coomassie brilliant blue R250.

2.3. Radioactive End-Labeling of a Purified DNA Restriction Fragment 1. Linear DNA fragment with convenient restriction sites on either end, previously phosphatased. 2. 10X T4 polynucleotide kinase buffer (supplied with enzyme). 3. [γ-32P]dATP, 6000 Ci/mmol. 4. T4 polynucleotide kinase 10,000 units/mL (Promega). 5. 2.5 M ammonium acetate. 6. 95% Ethanol, –20°C. 7. 70% Ethanol, –20°C. 8. 10% SDS stock solution. 9. Alkaline phosphatase from calf intestine (Boehringer Mannheim) 10. TE buffer: 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA.

2.4. In Vitro Reconstitution of Nucleosomes 1. 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA solutions containing 1.2 M, 1.0 M, 0.8 M, and 0.6 M NaCl. 2. TE buffer. 3. Stock of 6000–8000 molecular-weight cutoff dialysis tubing, 14 mm in diameter. 4. Stock of sonicated calf thymus (CT) DNA, approx 1–2 mg/mL. 5. Stock of 5 M NaCl. 6. Source of purified core histone proteins H2A/H2B and H3/H4, ours were purified from chicken erythrocytes (see Note 7).

2.5. Maxim–Gilbert G-Specific Reaction 1. 10X G-specific reaction buffer: 0.5 M NaCacodylate and 10 mM EDTA. 2. Dimethylsulfate (DMS) (Sigma). 3. G-reaction stop buffer: 1.5 M Na acetate, 1 M β-mercaptoethanol and 0.004 µg/µL sonicated calf thymus DNA. 4. Piperidine (neat, 10 M stock) (Sigma).

2.6. Chemical Mapping of Protein–DNA Interactions with EPD 1. 0.7% agarose made with 0.5X TBE. (Note: Treat all solutions with chelex 100 resin [Bio-Rad] to remove adventitious redox-active metals.) 2. Histone dilution buffer: 10 mM Tris-HCl, pH 8.0, and 50 mM NaCl. 3. Stock solution of 20 mM sodium ascorbate, store frozen at –20°C. 4. Stock solution of 1 mM Fe(II) 2 mM EDTA; store frozen at –20°C.


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5. Solution of 0.15% H2O2, freshly made. 6. Stop solution: 50% glycerol and 10 mM EDTA. 7. Microcentrifuge filtration devices (Series 8000 can be obtained from Lida Manufacturing Corporation). 8. 10 mM Tris-HCl, pH 8.0, and 0.1% SDS. 9. Microcentrifuge pestles, can be obtained from Stratagene. 10. 95% and 70% ethanol solutions, cooled to –20°C. 11. 3 M sodium acetate.

2.7. Sequencing Gel Analysis 1. 2. 3. 4. 5. 6.

Solid urea; molecular biology grade. 5X TBE. 40% Acrylamide (19:1 acrylamide:bis-acrylamide). 20% APS (ammonium persulfate). TEMED (N,N,N',N'-tetramethylethylenediamine). 1 mL formamide loading buffer: 100% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, and 1 mM EDTA.

3. Methods 3.1. Overexpression and Purification of Single-CysteineSubstituted Proteins The following methods work well for incorporating a single amino acid substitution into any protein of interest. Standard three or four primer PCR methods are used. 1. Standard PCR methods are used to amplify a DNA fragment containing a cysteine codon in place of the wild-type codon. If the codon to be changed is near the end of the amplified coding region, then only two primers are necessary with the change incorporated into one of these “parent” primers. If more central to the sequence, then a 3 primer technique is used with the change incorporated into an internal primer, amplify with the internal primer and one of the parent primers and then use the short amplified fragment as a primer with the remaining parent primer and the original DNA as the template. Finally, if this method fails, two complementary internal primers with the intended change are used to amplify overlapping short fragments using the appropriate parent primers; these two fragments are then combined with the parent primers and the entire insert amplified without an additional template added. 2. Gel purify the DNA insert of interest. We typically use the electroelution technique after separating the PCR DNA on a 1% agarose gel. A slice of agarose containing the insert of interest is placed into a dialysis membrane with enough TBE to cover the agarose. Both ends of the dialysis membrane are clipped shut and placed into a standard DNA electrophoresis apparatus containing 1X TBE buffer; the DNA electroeluted is for 15 min at 150 V. Remove the TBE buffer from the dialysis membrane containing the PCR DNA and precipitate.

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3. Ligate the insert containing the single cysteine substitution into the appropriate expression vector. We typically use the pET expression system (Novagen). Both DNAs must be digested with the same restriction endonucleases. Incubate equimolar amounts of insert DNA and pET3d DNA in 1X T 4 ligation buffer. Add 400 U of T4 DNA ligase (Bio-Labs) and incubate at 4°C overnight (see Note 2). 4. Check the efficiency of the ligation by transforming a small amount of the ligation sample into DH5a cells. Place the transformation on LB–ampicillin plates and incubate at 37°C overnight. 5. Prepare DNA from several colonies by placing a single colony into 3–5 mL of LB–ampicillin medium and grow at 37°C. Isolate the DNA from these cultures by standard DNA mini-prep techniques (see Note 3). 6. Digest part of the isolated plasmid with the original restriction endonucleases used for ligation to liberate the DNA fragment corresponding to the original insert. The plasmids that contain correct inserts can be used to transform BL21 cells for overexpression. 7. Transform the pET plasmid containing the insert into BL21 cells in the same manner as for the DH5a cells (see step 4). 8. Place one BL21 colony from the LB–ampicillin plate into 200 mL of LB–ampicillin medium. 9. Grow the culture in the absence of IPTG at 37°C to an optical density of 0.6 at 595-nm wavelength light. Add IPTG to a final concentration of 0.2 mg/mL and return the culture to 37°C for approx 2–4 h (see Note 4). 10. After 2–4 h, pellet the bacteria by centrifugation at 4000g for 15 min. 11. Decant the supernatant and resuspend the pellet in 5–10 mL of TE buffer. 12. Add 10 mg/mL lysozyme to a final concentration of 0.2 mg/mL. Then, add Triton-X 100 to a final concentration of 0.2% and incubate for 30 min at room temperature. 13. Dilute the bacteria twofold with 2 M NaCl to a final concentration of 1 M NaCl. Transfer the bacteria to Oakridge centrifuge tubes on ice. 14. Sonicate the bacterial slurry for 6 min total in two 3-min sonication steps (see Note 5). 15. Pellet the cell debris by centrifugation at 10,000g for 30 min at 4°C. 16. Add PMSF to a final concentration of 0.1 mM. Dilute the supernatants twofold with TE buffer to bring the NaCl concentration to 0.5 M. 17. Incubate the diluted supernatant with 12.5 mL of a 50% suspension of Bio-Rex 70-mesh beads for 4 h at 4°C with rotation. Linker histones and most other proteins will bind directly to the Bio-Rex 70 beads. However, core histone proteins must first be incubated with their dimerization partner proteins before they will bind to the chromatography matrix (i.e., H2A with H2B) (see Note 6). 18. After 4 h collect the beads in a plastic 10-mL disposable chromatography column. Collect the flowthrough fraction in a 50-mL conical tube and freeze. 19. Wash the column with 2–3 column volumes of 10 mM Tris-HCl, pH 8.0 containing 0.6 M NaCl. Collect the first 10 mL of the wash fraction in a 15-mL conical tube and freeze.


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20. Elute the bound proteins with two separate 1-column volume elution steps of 10 mM Tris-HCl, pH 8.0 containing 1.0 M NaCl. Collect the 1.0 M elution steps in separate 15 mL conical tubes and freeze. 21. After elution, wash the column with one column volume of 10 mM Tris-HCl, pH 8.0 containing 2.0 M NaCl. Collect the 2.0 M elution step in a 15-mL conical tube and freeze. 22. Check 10 µL of each fraction for protein by loading a small amount on a 12% or 18% SDS–polyacrylamide gel.

3.2. Reduction and Modification of Cysteine-Substituted Proteins 3.2.1. Reduction of Cysteine-Substituted Proteins 1. Incubate protein of interest in a 15-mL conical tube with 50 mM DTT final concentration for 1 h on ice. 2. Dilute the protein sample twofold with TE; this dilutes the NaCl concentration to 500 mM NaCl. 3. Add 0.8 mL of a 50% slurry of Bio-Rex 70 chromatography resin and incubate for 2 h at 4°C with rotation. We have found that Bio-Rex 70 can bind approx 0.5 mg protein/g resin. 4. Pour slurry into a 10-mL plastic, disposable chromatography column and collect the flowthrough fraction. Disposable chromatography columns are commercially available from Bio-Rad or other manufacturers. 5. Wash the column with 3–5 column volumes of buffer containing 10 mM Tris-HCl, pH 8.0, and 0.5 M NaCl. Immediately remove 20 µL of the freshly eluted wash sample into a separate Eppendorf tube for analysis later on a 12% SDS–polyacrylamide gel and immediately freeze the larger sample in case the protein did not bind the resin. Aliquoting the sample in this manner ensures that the sample does not need to be thawed for analysis. 6. An intermediate wash of the column with buffer containing 0.6 M NaCl is performed to remove proteins that are less well-bound because of partial degradation. Aliquots of these samples are obtained in the same manner as the previous wash step. 7. Linker histone protein can be eluted with 1-column volume steps of the same buffer except with 1.0 M NaCl. Typically, five separate 1-column volume 1.0 M NaCl elution steps are performed and collected separately. Usually, only 5 µL of the elution steps needs be aliquoted for SDS–polyacrylamide gel analysis. As above, the large elution fractions are frozen immediately. A final elution with buffer containing 2.0 M NaCl buffer will ensure that all of the protein has been eluted from the column. 8. Check the protein content of each aliquot obtained from the wash and elution steps on a 12% SDS–polyacrylamide gel. After separation, incubate the protein gel in enough Coomassie blue stain to cover the protein gel. Stain for approx 1 h at room temperature and destain with 45% methanol and 10% acetic acid until the background of the gel is clear.

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3.2.2. Modification of Cysteine Substituted Proteins with EPD 1. Thaw the fraction containing the reduced protein to be modified with EPD on ice. Working as quickly as possible, add a 1.1 fold molar excess of EPD to 60 µL (approx 30 µg) of reduced protein. Incubate for 1 h at room temperature in the dark. 2. Removal of excess cleavage reagent requires one more round of Bio-Rex 70 chromatography, identical to that in step 3 of Subheading 3.2.1. except that 60 µL of the 50% slurry is added to the protein. In addition, the slurry is poured into a column made from a blue 1-mL pipet tip fitted with glass wool at the opening. Wash and elute as in step 5 of Subheading 3.2.1. except all elution volumes are scaled according to the resin amount. Aliquots for protein analysis are exactly the same size as previously indicated. 3. Postmodification labeling with 14C-NEM (N-[ethyl–1–14C]-maleimide) (New England Nuclear) can be used to quantitatively determine the extent of modification with the DNA cleavage reagent. Add 0.25–0.5 µCi of 14C-NEM to each protein aliquot made from the elution fractions of the Bio-Rex column. After 10 min, add 2 volumes of 2X protein loading buffer and separate the proteins on a 12% SDS-polyacrylamide gel. Stain and destain the gel as in step 8 of Subheading 3.2.1. and dry the gel onto a piece of Whatman filter paper. Visualize the labeled proteins by exposing the dried gel to ultra sensitive Bio-Max autoradiography film. 4. A protein gel at this step performs two functions. (1) It determines which fractions contain the protein of interest and (2) it determine the extent of modification with the DNA cleavage reagent.

3.3. Radioactive End-Labeling of a Purified DNA Restriction Fragment 1. Treat approx 5 µg of plasmid DNA or 1 µg of a purified DNA fragment with the appropriate restriction endonuclease in the manufacturer’s buffer. 2. Precipitate the DNA by adjusting the solution to 0.3 M sodium acetate and adding of 2.5 vol of cold ethanol. 3. Resuspend the DNA in phosphatase buffer and treat with alkaline phosphatase for 1 h at 37°C. 4. Adjust the solution to 0.1% SDS, phenol extract the solution, and then precipitate the aqueous phase twice with ethanol and sodium acetate. 5. Resuspend the DNA in 10 µL TE and add 2.5 µL of 10X T 4 polynucleotide kinase buffer. 6. Add 50 µCi of [γ-32P]dATP and adjust the volume to 24 µL with water. 7. Start the reaction by adding 10 units of T4 polynucleotide kinase and incubate for 30 min at 37°C. 8. Stop the kinase with 200 µL of 2.5 M ammonium acetate (NH4Oac) and 700 µL of cold 95% ethanol. 9. Pellet the DNA in a microcentrifuge for 30 min at room temperature.


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10. Wash the DNA pellet briefly with cold 70% ethanol and dry the DNA in a Speedvac concentrator. 11. Dissolve the DNA in 34 µL of TE buffer. 12. Digest the DNA fragment with a second restriction endonuclease that liberates the fragment of interest and yields fragments that can be easily separated on a native 6% polyacrylamide gel. 13. After separation, wrap the gel tightly in plastic wrap and apply fluorescent markers onto various portions of the gel for alignment purposes (can be obtained from Stratagene) or accurately mark the position of the gel on the film. Expose the wet gel to the autoradiography film for 1 min, which is sufficient to detect the specific band containing the labeled fragment. 14. Excise the band of interest from the polyacrylamide gel and place into a clean Eppendorf tube. Crush the acrylamide gel slice with a Eppendorf pestle and add 700 µL of TE buffer. The labeled fragment will elute overnight with passive diffusion. 15. Split the sample equally into two Series 8000 Microcentrifuge Filtration Devices and spin for 30 min in a microcentrifuge. 16. Precipitate the eluted DNA and dissolve in TE buffer pH 8.0. Add enough TE buffer so that the labeled DNA is approx 1000 cpm/µL (see Note 7).

3.4. Reconstitution of Nucleosomes by Salt Step Dialysis The method described here for the reconstitution of nucleosomes allows for large quantities of nearly homogeneous core particles in 12 h (13). Moreover, reconstituted nucleosomes are known to bind linker histone in a physiologically relevant manner according to multiple criteria. Virtually any piece of DNA 147 bp or longer can be used. However to obtain nucleosomes with only one translational position, the DNA sequence should contain nucleosome positioning sequences such as that from the Xenopus borealis somatic 5S rRNA gene (14–16). The DNA can be labeled on the 5' or 3' end with commercially available enzymes after phosphatase treatment as described above. 1. Add approx 5–8 µg of unlabeled calf thymus DNA, 200,000–400,000 cpm of singly labeled Xenopus borealis 5S ribosomal DNA, purified chicken erythrocyte core histone protein fractions (H2A/H2B and H3/H4) (see Note 1), 160 µL of 5 M NaCl (2.0 M final), and TE buffer to a final of volume 400 µL. 2. Place the reconstitution mixture into a 6 to 8 kDa molecular weight cut-off dialysis bag. All subsequent dialysis steps are for 2 h at 4°C against 1 L of dialysis buffers unless specified. The first dialysis buffer is 10 mM Tris-HCl, pH 8.0, 1.2 M NaCl and 1 mM EDTA. Subsequent dialyses steps are with fresh buffer containing 1.0 M, 0.8 M, and then 0.6 M NaCl. The procedure is completed with a final dialysis against TE buffer overnight. Nucleosomes at this stage can be used for gel-shift experiments where EDTA does not interfere. 3. For DNA cleavage experiments with EPD, two additional dialysis steps are required. First dialyze the reconstitutes against 10 mM Tris-HCl, pH 8.0 several

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3.5. Maxim–Gilbert G-Specific Reaction The G-specific reaction used in the Maxim–Gilbert sequencing method provides an easy and quick method to identify the exact location of bases within any known sequence on sequencing gels. It is used here to determine the sites of DNA to base-pair resolution. Because this method is not generally used any longer, the steps are outlined as follows: 1. Add approx 20,000 cpm of singly labeled DNA (same DNA used to reconstitute nucleosomes). 2. Add 20 µL of 10X G-specific reaction buffer. 3. Add water to a final volume of 200 µL. 4. Start by adding 1 µL of straight dimethylsulfate (DMS) to the tube. Mix immediately and spin briefly in a microfuge (do this in a hood; be careful not to get any DMS on your skin or on standard laboratory gloves. Store DMS in a tightly capped brown glass bottle at 4°C). 5. Add 50 µL of G-reaction stop solution and mix immediately. 6. Add 2.5 vol of –20°C 95% ethanol to precipitate the DNA. 7. Wash the DNA with –20°C ethanol; dry and dissolve the DNA in 90 µL of H2O. 8. Add 10 µL of piperidine and incubate at 90°C for 30 min. 9. Dry the DNA solution in a Speedvac to completion. 10. Dissolve the DNA in 20 µL of water and repeat the drying step. Repeat this step one more time. 11. Dissolve DNA in 100 µL TE buffer and store at 4°C.

3.6. Site-Directed Hydroxyl Radical Cleavage of DNA 3.6.1. Binding Single-Cysteine-Substituted Linker Histone Proteins to Reconstituted Nucleosomes 1. The exact amount of each mutant linker histone protein needed to stoichiometrically bind the nucleosome needs to be determined empirically. Increasing amounts of the linker histone are titrated to a fixed volume of reconstituted nucleosomes (typically 5000 cpm) and analyzed via a gel-shift procedure (17). This is typically scaled up 10-fold for the site-specific cleavage reaction. 2. Add 5% glycerol final to the binding reaction (analytical scale only, site-specific cleavage reactions contain 10-fold less glycerol). 3. Add 50 mM NaCl final to the binding reaction (see Note 8). 4. Incubate the binding reactions for 15 min at room temperature. 5. Separate the complexes on a 0.7% agarose and 0.5X TBE gel. After drying the gel, expose to autoradiograpic film and determine the amount of protein necessary for good complex formation.


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6. Several assays for the correct binding of linker histones to DNA have been performed (17,18). One of the easiest involves a brief digestion with micrococcal nuclease in the chromatosome stop assay (13).

3.6.2. Site-Directed Hydroxyl Radical Mapping of Linker Histone-DNA Interaction 1. Scale up the binding reaction to include 40,000–50,000 cpm of labeled reconstituted nucleosomes and add enough modified mutant linker histone to form H1–nucleosome complexes. 2. Add glycerol to 0.5% final concentration (see Note 9). 3. Add sodium ascorbate to a final concentration of 1 mM. 4. Add H2O2 to a final concentration of 0.0075%. 5. Incubate for 30 min at room temperature in the dark. 6. After 30 min, add 1/10 vol of 50% glycerol and 10 mM EDTA solution. 7. Load samples immediately onto a running (90 V) preparative 0.7% agarose and 0.5X TBE gel. 8. Separate the samples so that the H1–nucleosome complexes are well resolved from tetramer and free DNA bands. 9. Next, wrap the gel tightly with plastic wrap so that the gel cannot move within the plastic. Lay fluorescent markers onto various portions of the gel for alignment purposes (can be obtained from Stratagene) or accurately mark the position of the gel on the film. 10. Expose the wet gel for several hours at 4°C. 11. Next, develop the autoradiograph and overlay onto the wet gel, lining up the fluorescent markers. 12. Cut and remove the agarose containing the H1–nucleosome complexes or bands of interest and place them into Series 8000 Microcentrifuge Filtration Devices. 13. Freeze the filtration tubes containing the agarose plugs on dry ice for 15 min. 14. Spin down the agarose in a microcentrifuge at maximum speed for 30 min at room temperature. The fluid from the agarose matrix will be collected in the 2-mL centrifuge tube surrounding the filtration device. 15. Gently remove the agarose plug from the bottom of the filtration device and place into a clean Eppendorf tube. Save the centrifugation devices for use later. 16. Using a microcentrifuge pestle, crush the agarose pellet and add 500 µL of 10 mM Tris-HCl, pH 8.0, and 0.1% SDS and continue to crush the agarose. 17. After the agarose is crushed into tiny pieces, place all samples at 4°C overnight. 18. Place the crushed agarose into the same centrifugation device and pellet. Spin down the agarose in a microcentrifuge at maximum speed for 30 min at room temperature. 19. Combine identical samples from both spins and precipitate the DNA. 20. Dissolve the DNA in 15 µL of TE buffer. 21. Heat the samples to 90°C for 2 min to denature.

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3.7. Sequencing Gel Analysis of H1°C-EPD Cleavage 3.7.1. Sequencing Gel Electrophoresis 1. Add equal numbers of counts from each sample, including the G specific reaction, to clean eppendorf tubes. 2. Place the samples into a Speedvac concentrator and dry to completeness. 3. Dissolve the samples in 4 µL of formamide loading buffer. 4. Place samples directly onto ice to prevent renaturation. 5. Separate samples on a 6% polyacrylamide and 8 M urea sequencing gel running at constant 2000 V.

3.7.2 Example of Site-Directed Cleavage of Nucleosomal DNA by EPD An example of a linker histone site-directed DNA cleavage reaction is presented in Fig. 2. A schematic of the 5S mononucleosome is shown in the left panel. The thick black line represents the 5S ribosomal DNA fragment that contains the transcriptional coding sequence for this gene (gray arrow). The 5S ribosomal gene fragment was used because it contains a nucleosomal positioning sequence that precisely wraps the DNA around the core histones and provides a homogeneous population of nucleosomes. Furthermore, because one major translational position predominates within this population of nucleosomes, the precise orientation of the DNA as it wraps around the core histones is known. This enables us to determine to base-pair resolution, the sites of cleavage by EPD with respect to the nucleosome structure. A singly endlabeled 5S DNA fragment was incorporated into nucleosomes via the salt dialysis procedure detailed earlier. Labeled mononucleosomes were bound by an EPD-modified linker histone containing a single cysteine substitution for the lysine residue at position 59, referred to as K59C-EPD. After allowing hydroxyl radical cleavage for 30 min, the protein–DNA complexes were separated on a 0.7% agarose gel (Fig. 2A) and the labeled DNA fragments corresponding to the H1–nucleosome complexes were purified. These purified DNAs were then analyzed on a 6% sequencing gel (Fig. 2B, right panel).

Fig. 2 (opposite page). (A) Various DNAs from control or hydroxyl radical cleavage reactions were separated on a 0.7% agarose gel. The wet gel was exposed to autoradiographic film for 3–4 h according to Subheading 3.6.2. Lanes 1 and 2 contain free DNA (FD) and bulk nucleosomes (Nuc.), respectively, not exposed to hydroxyl radical cleavage. Lanes 3–5 contain nucleosomes or H1–nucleosome complexes (H1–Nuc.) subjected to hydroxyl radical cleavage. Nucleosomes–K59C (lane 3), nucleosomes with unmodified K59C (lane 4) or nucleosomes with K59C–EPD (lane 5). (B) A linear schematic of the 5S nucleosome is shown (left). The DNA (black line)


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was restricted with the restriction enzymes shown and radiolabeled (*) at the XbaI site. The position of the 5S nucleosome (white oval) is shown with respect to the start site of transcription (gray arrow) and with respect to the size of DNAs in the sequencing gel (larger white oval). Labeled DNAs from hydroxyl radical cleavage reactions were analyzed on a 6% sequencing gel (right). Lane 1: Maxim–Gilbert G-specific reaction; lane 2: hydroxyl radical cleavage in the absence of histone H1; lanes 3 and 4: hydroxyl radical cleavage with K59C (lane 3) or K59C–EPD (lane 4). Specific cleavages are shown to the right of the gel (black arrows).

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The gel reveals that the reconstituted mononucleosomes used for this experiment give a characteristic 10-bp protection when footprinted by general cleavage with hydroxyl radical in the absence of linker histone (15). This indicates that the 5S DNA has been properly assembled with the histone octamer into a nucleosome (Fig. 2B, lane 2). The linker histone-directed cleavage experiments are shown in lanes 3 and 4. No cleavages are observed when the reaction is carried out in the presence of unmodified K59C (Fig. 2B, lane 3). In contrast, when the cleavage reagents are added in the presence of K59C-EPD bound nucleosomes, two sets of cleavages are evident (Fig. 2B, lane 4). The cleavages at +62, +72, and +82 correspond to the end of the nucleosome where the DNA exits. This result is consistent with previous data suggesting that the linker histone binds the nucleosome at the periphery, tucked inside a superhelical gyre of DNA (12,19). A second set of cleavages occurs at –29 and –39. These cleavages occur on the DNA strand directly underneath that of the +62/ +72/+82 cleavages as the DNA makes one full superhelical turn around the histone octamer. It is possible that amino acid 59 makes close contacts with both strands of the DNA, consistent with the strong cleavages seen at each site. It is also possible that hydroxyl radicals have diffused away from the –29/–39cleavage site and cleave the DNA in other areas. Inconsistent with this, glycerol, a very good hydroxyl radical scavenger, does not seem to have an effect on the cleavages obtained with K59C-EPD at concentrations known to eliminate hydroxyl radical cleavage (Chafin and Hayes, unpublished results). 4. Notes 1. A complication of the in vitro reconstitution procedure is that purified histone proteins are often obtained in two fractions, H2A/H2B and H3/H4 (22). Thus, in addition to total histone mass, the ratio between these two substituents must be empirically adjusted to yield maximum octamer–DNA complexes (13). 2. Many ligation procedures are available from primary literature or commercial sources. Ligation of two DNA fragments occurs more rapidly at room temperature or 37°C if the base-pair overlap is sufficiently stable. 3. Many DNA mini-prep procedures are described in detail in ref. (20). The DNA isolated for the techniques described here were from the boiling DNA mini-prep procedure (20). 4. Before proceeding, it is recommended that a small amount of the culture be checked for overexpression of the protein of interest. This can be done by removing 1 mL of the culture before and after induction by IPTG. 5. Sonication techniques tend to increase the temperature of the sample quickly, which could induce proteolysis of the proteins. The sample must therefore be cooled before and during sonication. Allow several min between sonication runs to keep the sample as cold as possible. 6. Histones H2A or H2B do not bind to Bio-Rex 70 when purified individually. However, we have found that when allowed to heterodimerize, they bind to the


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column and elute off consistently in 1 M NaCl (13). This characteristic could be the result of the fact that histone H2A and H2B are completely unfolded when separated from each other (21). 7. Storing labeled DNA in a concentrated form is not advised, as autodegradation of the DNA takes place. DNA can be stored for several weeks at approx 5000 cpm/µL. 8. Several methods can be used for the incorporation of linker histones into reconstituted mononucleosomes. The method described here involves direct addition of linker histones to mononucleosis in 50 mM NaCl. Linker histones are folded in low-salt solutions in the presence of DNA (23). Indeed, we find that linker histones can be directly mixed to nucleosomes in either 5- or 50-mM NaCl solutions and these proteins then bind in a physiologically relevant manner (17). 9. Glycerol is a good scavenger for hydroxyl radicals and will generally inhibit hydroxyl-radical-based cleavage if added at a final concentration over 0.5% and therefore should be avoided. However, adding small concentrations of glycerol will allow hydroxyl radical cleavage to occur if the EPD moiety is in close proximity to the DNA backbone but not cleavage from sites farther away.

References 1. Wolffe, A. P. (1995) Chromatin: Structure and Function. Academic, London. 2. van Holde K. E. (1989) Chromatin. Springer-Verlag, New York. 3. Thoma, F., Koller, T., and Klug, A. (1979) Involvement of histone H1 in the organization of the nucleosome and the salt-dependent superstructures of chromatin. J. Cell Biol. 83, 403–427. 4. Carruthers, L. M., Bednar, J., Woodcock, C. F. L., and Hansen, J. C. (1998) Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order folding. Biochemistry 37, 14,776–14,787. 5. Crane-Robinson, C. (1997) Where is the globular domain of linker histone located on the nucleosome? Trends Biochem. Sci. 22, 75–77. 6. Zhou, Y.-B., Gerchman, S. E., Ramakrishnan, V., Travers, Andrew, and Muyldermans, S. (1998) Position and orientation of the globular domain of linker histone H5 on the nucleosome. Nature 395, 402–405. 7. Ebright, Y. W., Chen, Y., Pendergrast, P. S., and Ebright, R. H. (1992) Incorportation of an EDTA–metal complex at a rationally selected site within a protein: application to EDTA–iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro. Biochemistry 31, 10,664–10,670. 8. Ermacora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., and Fox, R. O. (1992) Conformation-dependent cleavage of staphlyococcal nuclease with a disulfide-linked iron chelate. Proc. Natl. Acad. Sci. USA 89, 6383–6387. 9. Neelin, J. M., Neelin, E. M., Lindsay, D. W., Palyga, J., Nichols, C. R., and Cheng, K. M. (1995) The occurrence of a mutant dimerizable histone H5 in Japanese quail erythrocytes. Genome 38, 982–990. 10. Chen, Y. and Ebright, R. H. (1993) Phenyl-azide-mediated photocrosslinking analysis of Cro–DNA interaction. J. Mol. Biol. 230, 453–460.

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11. Lee, K.-M. and Hayes, J. J. (1997) The N-terminal Tail of Histone H2A Binds to Two Distinct Sites Within the Nucleosome Core. Proc. Natl. Acad. Sci. USA 94, 8959–8964. 12. Hayes, J. J. (1996) Site-directed cleavage of DNA by a linker histone–Fe(II)EDTA conjugate: localization of a globular domain binding site within a nucleosome. Biochemistry 35, 11,931–11,937. 13. Hayes, J. J. and Lee, K.-M. (1997) In vitro reconstitution and analysis of mononucleosomes containing defined DNAs and proteins. Methods 12, 2–9. 14. Rhodes, D. (1985) Structural analysis of a triple complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA. EMBO J. 4(13A), 3473–3482. 15. Hayes, J. J., Tullius, T. D., and Wolffe, A. P. (1990) The structure of DNA in a nucleosome. Proc. Natl. Acad. Sci. USA 87, 7405–7409. 16. Simpson, R. T. (1991) Nucleosome positioning: occurrence, mechanisms and functional consequences. Prog. Nucleic Acids Res. Mol. Biol. 40, 143–184. 17. Hayes, J. J. and Wolffe, A. P. (1993) Preferential and asymmetric interaction of linker histones with 5S DNA in the nucleosome. Proc. Natl. Acad. Sci. USA 90, 6415–6419. 18. Allan, J., Hartman, P. G., Crane-Robinson, C., and Aviles, F. X. (1980) The structure of histone H1 and its location in chromatin. Nature 288, 675–679. 19. Pruss, D., Bartholomew, B., Persinger, J., Hayes, J. J., Arents, G., Moudrianakis, E. N., et al. (1996) A new model for the nucleosome: a binding site for linker histone inside the DNA gyre. Science 274, 614–617. 20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 21. Karantza, V., Baxevanis, A. D., Freire, E., and Moudrianakis, E. N. (1995) Thermodynamic studies of the core histones: Ionic strength and pH dependence of H2A–H2B dimer stability. Biochemistry 34, 5988–5996. 22. Simon, R. H. and Felsenfeld, G. (1979) A new procedure for purifing histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxyapatite. Nucleic Acids Res. 6, 689–696. 23. Clark, D. J. and Thomas, J. O. (1986) Salt-dependent co-operative interaction of histone H1 with linear DNA. J. Mol. Biol. 187, 569–580.

Nitration of Tyrosine Residues

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20 Nitration of Tyrosine Residues in Protein–Nucleic Acid Complexes Simon E. Plyte 1. Introduction Chemical modification is a powerful tool for investigating the accessibility and function of specific amino acids within folded proteins. It has provided significant information regarding the role of different amino acids at the binding sites of numerous enzymes and DNA-binding proteins. The identification of such residues by chemical modification has then often be used to plan subsequent site-directed mutagenesis experiments. These data complement those from crystallographic and nuclear magnetic resonance (NMR) studies in determining the residues located at the active site; thus, one needs to consider all these techniques when elucidating protein structure and function. For example, chemical modification of leukotriene A4 hydrolase, 3-hydroxyisobutyrate dehydrogenase, and lactate dehydrogenase (1–3) have contributed significantly to the understanding of active-site mechanisms in these proteins and in elucidating the mechanisms of DNA binding in the Fd and Pf1 gene 5 proteins (4–5). Reagents exist to modify cysteine, methionine, histidine, lysine, arginine, tyrosine and carboxyl groups selectively. However, in this chapter, we are only concerned with the selective modification of tyrosine residues (for reagents and conditions for the modification of the other amino acids, see ref. 6). The side chain of tyrosine can react with several compounds, the most commonly used being N-acetylimidizole and tetranitromethane (TNM). N-acetylimidizole will O-acetylate tyrosine residues in solution (7), and this reagent has been used to modify numerous proteins including the Fd gene 5 protein (4). However, this reagent can also N-acetylate primary amines, and in the study on the Fd gene 5 protein (4) in addition to acetylation of three tyrosine residues, all From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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five lysine residues were found to be modified. Tetranitromethane is a reagent highly specific for tyrosine residues and reacts under mild conditions to form the substitution product 3-nitrotyrosine (8). The modified tyrosine has a characteristic adsorption maximum at 428 nm, and this can be used to quantitate the number of tyrosine residues modified (8). However, under harsher conditions, there have been some reports of modification of sulfhydryl groups and limited cases of reaction with histidine and tryptophan (9).

1.1. Strategies 1.1.1. Tyrosine Accessibility The general strategy employed in chemical modification experiments is to determine the accessibility of the target residues within the native protein and the extent of protection offered by the bound substrate. Peptide mapping of the labeled protein then allows the roles of the individual residues to be assessed. First, the free protein is nitrated and then digested into fragments by proteolysis. These peptides are then separated to enable identification of the modified residue(s). The nucleoprotein complex is also nitrated and the modified residues identified in a similar way. From a comparison of these data, the extent of protection at each site can be established. For peptide mapping, a protease should be chosen that, on digestion of the target protein, will place each tyrosine in a separate peptide. However, this is not essential if the modified residues are identified by N-terminal sequencing. It is possible that tyrosine modification may affect the efficiency of α-chymotrypsin digestion and this enzyme should be avoided if possible. The peptides can be separated by reverse-phase high-performance liquid chromatography (HPLC), and those containing tyrosine purified for further analysis. The tyrosine-containing peptide can be easily identified directly after HPLC purification by the characteristic fluorescence emission maximum of 3-nitrotyrosine at 305 nm (when excited at 278 nm). A particular tyrosine residue can then be identified by N-terminal sequence analysis. The identification of nitrated tyrosine residues in the free protein provides information concerning the solvent accessibility of these residues in the protein and indicates which residues are likely to be buried within the protein. DNA protection studies will indicate which of these residues may be involved in protein–DNA interactions. However, the protection from nitration by bound DNA is only an indication of a functional role for a particular residue. The bound DNA may confer protection to a residue several angstroms away or may induce protein oligomerization (cooperative binding) which protects the tyrosine by protein–protein interactions. Consequently, functional studies need to be performed to further determine the role of the protected residue(s). The


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situation is analogous to the two types of analysis frequently used in the investigation of the DNA bases involved in complexes: “footprinting” and “interference” techniques. The data obtained from chemical modification and protection studies can then be used to design site-directed mutagenesis experiments to look at the function of an individual residue by observing the effects of its replacement with other amino acids.

1.1.2. Functional Studies A protocol for functional studies will not be described in this chapter, but some general considerations will be mentioned here. One should nitrate the free protein and determine whether the modified protein still binds to DNA. This information should indicate whether the residues protected in the nucleoprotein complex are implicated in DNA binding. However, with proteins that bind cooperatively to DNA, a reduction in DNA-binding affinity may result from a disruption of protein–protein interactions rather than from protein–DNA interactions. A possible way to resolve this ambiguity is to bind the native and modified proteins to short oligonucleotides where the cooperativity factor is negligible. Modification of residues involved in protein–protein interactions should not significantly affect the intrinsic binding of the modified protein to DNA, when compared to the native protein. Tyrosine residues can interact with DNA either by hydrophobic interactions via stacking with DNA bases or by hydrogen-bonding with the nucleotide through the phenolic OH group (10). Nitrotyrosine has a pKa of 8.0, which may disrupt H-bonding as well as base stacking interactions. However, the addition of sodium dithionate reduces 3-nitrotyrosine to 3-amino tyrosine (which has a pKa similar to that of native tyrosine) and may restore H-bonding interactions (11). Reduction with this reagent may provide further information concerning the nature of the tyrosine–nucleic acid interaction.

1.1.3. Rates of Modification Nitration of a protein will initially report on the accessibility of specific tyrosine residues in the presence and absence of DNA. However, if the modified tyrosine residues can be analyzed individually, one can look at the nitration rates of the tyrosines and determine the degree of accessibility of each residue. This is achieved by removing aliquots of protein (at various time intervals) from a nitration experiment and determining the percentage nitration of each tyrosine for a given time-point. This can be done by quantitating the nitrated and unnitrated products after digestion, either by measuring the peak areas (recorded at 214 nm), taken directly from the HPLC profile (5), or by amino acid analysis of the purified peptides.

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2. Reagents All chemicals should be of AnalaR grade or higher and dissolved in doubledistilled water. For HPLC analysis, trifluoroacetic acid (TFA), water and acetonitrile should be of HPLC grade. Buffers for HPLC should be filtered (0.2 µm) and degassed before use. 1. Tetranitromethane (TNM) stock solution: a 300 mM stock solution of TNM in ethanol. Store in the dark at 4°C. Note that TNM can cause irritation to the skin and lungs, and the solution should be made up in the fume hood. Additionally, TNM can be explosive in the presence of organic solvents such as toluene. 2. Nitration buffer: 150 mM NaCl and 10 mM Tris, pH 8.0. 3. Desalting column: Disposable “10DG” Econo columns (Bio-Rad, Richmond, CA) are preferred. 4. µBondapak C18 HPLC column (Waters Associates, Milford, MA) or a similar reverse-phase column. 5. Trypsin (TPCK treated). 6. Standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) equipment with DC power supply capable of 150 V. 7. SDS–polyacrylamide gel stock solutions: Solution A: 152 g acrylamide and 4 g bis-acrylamide. Make up to 500 mL. Solution B: 2 g SDS and 30 g Tris base, pH 8.8. Make up to 500 mL. Solution C: 2 g SDS and 30 g Tris base, pH 6.8. Make up to 500 mL. When making up these solutions, they should all be degassed and filtered using a Buchner filter funnel. They should be stored in lightproof bottles; they will keep for many months. 8. 10% ammonium persulfate (APS): dissolve 0.1 mg in 1 mL of dH2O. 9. 15% SDS–polyacrylamide gel: Mix together 8.0 mL of solution A, 4.0 mL of solution B, and 3.9 mL of dH2 O. Add 150 µL of 10% APS and 20 µL of N,N,N',N'-tetramethylethylene diamine (TEMED). Mix well and then pour between the plates. Immediately place a layer of dH2O (or butanol) on top of the gel to create a smooth interface with the stacking gel. When the resolving gel has set, pour off the water and prepare the stacking gel. This is done by adding 750 mL of solution A and 1.25 mL of solution C to 3.0 mL of dH2O. Finally, add 40 µL of APS and 10 µL of TEMED, pour on the stacking gel and insert the comb. To avoid the gel sticking to the comb, remove the comb as soon as the gel has set. 10. 10X SDS running buffer: 10 g SDS, 33.4 g Tris base, and 144 g glycine made up to 1 L. 11. High methanol protein stain: Technical-grade methanol 500 mL, 100 mL glacial acetic acid and 0.3 g PAGE 83 stain (Coomassie blue), made up to 1 L. 12. Destain solution: 100 mL methanol and 100 mL glacial acetic acid, made up to 1 L. 13. 2X SDS-PAGE loading buffer: 4% (w/v) SDS, 60 mM Tris-HCl, pH 6.8, 20% glycerol, 0.04% (w/v) bromophenol blue, and 1% (v/v) β-mercaptoethanol.


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3. Methods The method is a fairly general one for protein-nucleic acid complexes. However, precise details of conditions for nucleoprotein complex dissociation and peptide mapping will vary with the system under investigation. As an example of the technique, nitration of the Pf1 gene 5 protein and nucleoprotein complex will be described (5).

3.1. Nitration 1. Desalt the protein or nucleoprotein complex into nitration buffer to a concentration between 0.5 and 5 mg/mL (see Note 1). For initial determination of nitrated residues, 0.5 mg of protein should be sufficient. However, if a time-course experiment is performed, larger amounts of protein are required. 2. To 1 mL of sample, add a 10-fold molar excess of 300 mM TNM (in ethanol) and incubate at room temperature for 1 h, stirring gently (see Note 2). The reaction is stopped by the addition of acid (add HCl to pH 2.0) or by rapid desalting into 10 mM Tris–HCl pH 8.0 (see Note 3). 3. Run an aliquot of the modified protein–nucleoprotein complex on an SDS gel, together with native protein, to determine whether there has been any TNMinduced crosslinking (see Note 4). If analyzing the free protein proceed to step 5; if modifying the nucleoprotein complex, proceed to step 4. 4. Dissociate the nucleoprotein complex by the addition of salt (see Note 5). Large DNA fragments can be removed by ultracentrifugation, whereas smaller fragments can be either digested with nucleases or removed by gel filtration. The protein is then dialyzed or desalted into the appropriate protease digestion buffer. 5. Digest the protein to completion with the desired protease(s) and then lyophilize the peptides for separation by HPLC. The peptides may be stored at –20°C.

In the example provided, the Pf1 gene 5 protein and nucleoprotein complex were incubated at room temperature in the presence of a 64 M excess of TNM (300 mM in ethanol) for 3 h. The reaction was stopped by desalting the protein (and nucleoprotein complex) into 10 mM Tris-HCl, pH 8.0. The nucleoprotein complex was dissociated by the addition of MgCl2 to 1 M and the phage genomic DNA was then removed by ultracentrifugation at 221,000g (in a Beckman L8 ultracentrifuge; 70.1 Ti rotor) for 2.5 h. The protein was desalted into 10 mM Tris-HCl, pH 8.0 for proteolysis and digestion with trypsin (Sigma [St. Louis, MO], TCPK treated) at an enzyme substrate ratio of 1:25 (w/w) for 3 h at 37°C. Phenylmethane sulfonyl fluoride was added to a final concentration of 1 mM and the sample was lyophilized overnight. This procedure results in the complete separation of the three tyrosine-containing tryptic peptides.

3.2. Peptide Mapping 1. Peptides can usually be separated by reverse-phase HPLC on a C 18 column. Generally the peptides are applied to the column in 8 M urea and 2% β-mercapto-

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ethanol and separated in an acetonitrile gradient in the presence of 0.05–0.1% TFA. The acetonitrile gradient must be determined empirically for each particular protein. 2. Determine separation conditions for peptides from the native protein (see Note 6) and identify tyrosine-containing peptides (see Note 7). 3. Apply peptides from the nitrated protein and initially elute under the same conditions that were used for the native protein (see Note 8). If necessary, change the acetonitrile gradient to achieve separation of the tyrosine-containing peptides and their nitrated counterparts.

In the example provided, the tryptic peptides from a native gene 5 protein were resuspended in 200 µL of 8 M urea and 2% β-mercaptoethanol and clarified prior to HPLC analysis (Fig. 1, top). Tyrosine-containing peptides were initially detected by their fluorescence properties (see Note 7) and then identified by automated Edman degradation on an Applied Biosystems 477A pulsed liquid amino acid sequencer. Nitrated peptides were applied to the C18 column and separated under identical conditions (Fig. 1, bottom). The nitrated peptides were initially detected by their altered retention times and by virtue of their yellow color in 10 mM Tris-HCl, pH 8.0. The identity of the nitrated peptides was subsequently confirmed by N-terminal sequencing (see Note 9).

3.3. Functional Studies As discussed in Subheading 1., one should check whether nitration of the protein impairs the ability to bind DNA (other chapters in this volume can be consulted for possible approaches such as EMSA [Chapter 2] or DNaseI footprinting [Chapter 3]). The protein isolated from the nitrated nucleoprotein complex should be checked for DNA binding. Because the target amino acid residues in contact with the DNA should have been protected from modification, the protein from the nitrated complex would be expected to retain DNAbinding activity. 4. Notes 1. As an alternative to desalting, the protein can be dialyzed into nitration buffer. 2. The molar excess of TNM can be increased to ensure maximal modification (e.g., the Pf1 gene 5 protein was nitrated in a 64-fold molar excess of TNM in the example provided). Note, however, that at high concentrations, protein insolubility can become a problem. 3. One can desalt the protein into a buffer appropriate for proteolysis or dissociation of the nucleoprotein complex at this stage, as required. 4. Tetranitromethane-induced crosslinking has been widely reported, and an SDS gel should be run to check for the appearance of adducts. Reducing the concentration of portion or molar excess of TNM may help to limit adduct formation.


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Fig. 1. High-performance liquid chromatographic elution profile of tryptic peptides of the Pf1 gene 5 protein for (top) native protein and (bottom) nitrated protein. Peaks Y21, Y30, and Y55 correspond to tryptic peptides containing tyrosine 21, 30, and 55, respectively. n denotes a nitrated peptide. Aliquots (100 µL) were applied to a µBondapak C18 HPLC column (Waters Associates) (300 × 4.6 mm inner diameter) fitted with a C18 guard column. The HPLC buffers for this experiment were as follows: buffer A: 0.05% TFA/H2O; buffer B: 0.05% TFA/acetonitrile. Peptides were separated in the following gradient at a flow rate of 2 mL/min: O% B for 5 min; 0–10% B in 20 min; 10–55% B in 45 min; 55–90% B in 5 min; 90% B for 5 min; 90–100% B in 5 min.

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5.

6.

7.

8.

9.

10.

Plyte Gel filtration is another way of removing the adducts prior to peptide mapping (see also Note 10). Usually, protein–nucleic acid interactions can be disrupted by the addition of NaCl or MgCl2 to 1–2 M. The conditions required to effect separation will vary with the nature of the complex. It is not essential to have complete separation of all fragments, only separation of the tyrosine-containing peptides and their nitrated counterparts. The HPLC conditions should be adjusted to achieve this. Tyrosine residues have a characteristic fluorescence emission maximum at 303 nm when excited at 278 nm. This phenomenon can be used initially to determine which peptides contain a tyrosine residue (this may not be possible, however, if there is a tryptophan residue present in the same peptide as a result of energy transfer). If on-line fluorescence detection is not available, the fractions can be taken directly from the HPLC and analyzed in a fluorimeter. The peptides should then be unambiguously identified by either N-terminal sequencing or amino acid analysis. The addition of a nitrate group to the tyrosine should alter the hydrophobicity and, hence, the retention time of that particular peptide in an acetonitrile gradient. This should allow immediate identification of the nitrated peptides. However, it is possible that a nitrated peptide comigrates with another unmodified peptide. Therefore, freeze-dry all peptides from HPLC and resuspend in 10 mM Tris-HCl, pH 8.0: The nitrated peptides will have a faint yellow color (absorbance maximum at 428 nm). For peptides sequenced on an applied biosystems 477A pulse liquid amino acid sequencer (fitted with a 120A separation system for the analysis of PTH-derivitized amino acids), PTC-3-nitrotyrosine elutes just after DTPU. TNM induced oligomerization has been observed on the nitration of numerous proteins including several DNA-binding proteins (4,12,13). This is usually considered undesirable, and steps are often taken to reduce the crosslinking and remove adducts before analysis (e.g., by gel filtration). However, advantage can be taken of this crosslinking ability; Martinson and McCarthy (14) used TNM as a reagent to crosslink histones specifically. On nitration with TNM, we have also shown that the Pf1 gene 5 protein forms an SDS-stable dimer (13). Initial analysis of the peptide adduct in this case suggested that tyrosine 55 from one monomer was crosslinked to phenylalanine 76 from the other monomer (forming an interdimer crosslink rather than an intradimer crosslink). The adducts are thought to form via a free-radical mechanism, resulting in zero-length crosslinks between residues in close proximity (14,15) Thus, if adduct formation is limited to one or two species, additional structural information can be obtained from the experiment. The crosslinked proteins should be digested and the peptide adduct purified by HPLC. N-Terminal sequencing, amino acid analysis, and gas chromatograpy–mass spectroscopy (of the hydrolyzed peptide) should enable unambiguous identification of the two residues participating in the crosslink and provide structural information concerning the relative positions of these residues in the protein.


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References 1. Mueller, M. J., Samuelson, B., and Haeggstrom, J. Z. (1995) Chemical modification of leukotriene A4 hydrolase. Indications for essential tryosyl and arginyl residues at the active site. Biochemistry 34, 3536–3543. 2. Hawes, J. W., Crabb, D. H., Chan, R. M., Rougraff, P. M., and Harris, A. (1994) Chemical modification and site-directed mutagenesis studies of rat 3-hydroxyisobuyrate dehydrogenase. Biochemistry 34, 4231–4237. 3. Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P., and Hottinger, H. (1992) Primary strucure, physiochemical properties and hemical modification of NAD+dependent d-lactate dehydrogenase. J. Biol. Chem. 267, 8499–8513. 4. Anderson, R., Nakashima, Y., and Coleman, J. (1975) Chemical modification of functional residues of the Fd gene 5 DNA-binding protein. Biochemistry 14, 907–917. 5. Plyte, S. E. and Kneale, G. G. (1991) Mapping the DNA binding site of the Pf1 gene 5 protein. Protein Eng. 4(5), 553–560. 6. Lundblad, R. and Noyes, M. (1984) Chemical Reagents for Protein Modification I and II, CRC, Boca Raton, FL. 7. Riordan, J., Sokolovsky, M., and Vallee, B. (1967) Environmentally sensitive tyrosine residues. Nitration with tetranitromethane. Biochemistry 6, 358. 8. Sokolovsky, M., Riordan, J., and Vallee, B. (1966) Tetranitromethane. A reagent for the nitration of tyrosyl residues in proteins. Biochemistry 5, 3582–3589. 9. Sokolovsky, M., Harell, G., and Riordan, J. (1969) Reaction of tetranitromethane with sulphydryl groips in proteins. Biochemistry 8, 4740–4745. 10. Dimicoli, J. and Helene, C. (1974) Interaction of aromatic redisues of proteins with nucelic acids I and II. Biochemistry 13, 714–730. 11. Sokolovsky, M. Riordan, J., and Vallee, B. (1967) Conversion of 3-nitrotyrosine to 3-amino-tyrosine in peptides and proteins. Biochem. Biophys. Res. Commun. 27, 20. 12. Anderson, R. and Coleman, J. (1975) Physiochemical properties of DNA-binding proteins: gene 32 protein of T4 and Escherichia coli unwinding protein. Biochemistry 1, 5485–5491. 13. Plyte, S. E. (1990) The biochemical and biophysical characterization of the Pf1 gene 5 protein and its complex with nucelic acids, Ph.D. thesis, Portsmouth University, Portsmouth, UK. 14. Martinson, H. and McCarthy, B (1975) Histone–histone associations within chromatin. Crosslinking studies using tetranitromethane. Biochemistry 14, 1073–1078. 15. Williams, J. and Lowe, J. (1971) The crosslinking of tyrosine with tetranitromethane. Biochem. J. 121, 203–209.

Modification of Lysine by Reductive Methylation

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21 Chemical Modification of Lysine by Reductive Methylation A Probe for Residues Involved in DNA Binding Ian A. Taylor and Michelle Webb 1. Introduction The basic side chains of lysine residues often play essential roles in DNA– protein recognition. They are able to contribute to the overall affinity of an interaction through nonspecific charge–charge interactions with the phosphate backbone and contribute substantially to the specificity of the interaction by forming direct hydrogen bonds with functional groups on the edges of the bases. This dual role and their almost ubiquitous presence in the interface of DNA–protein complexes make them very attractive targets for chemical modification experiments. Numerous chemical reagents to chemically modify lysine side chains in proteins are available (1). Unfortunately, the conditions required for such procedures are often harsh and result in total denaturation of the protein. Furthermore, many reagents are not entirely specific to lysine and often react with other residues such as cysteine, histidine, and tyrosine. Two methods, that are able to specifically modify lysine residues under native conditions and have been applied successfully to the investigation of protein nucleic acid interactions, are amidination with imidoesters (2) and, the subject of this chapter, reductive alkylation (3). Reductive alkylation has become a widespread and well-established technique for the specific modification of lysine residues and a variety of reagents have been used to produce this chemical modification (4). A reductive methylation reaction using formaldehyde and the reducing agent sodium cyanoborohydride (5) is particularly useful because under mild solution condiFrom: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Fig. 1. Reductive methylation of lysine. The reaction scheme (6) occurs in two distinct phases. Adduction of formaldehyde to the lysine ε-amino group generates the Schiff base (A), which is then reduced by sodium cyanoborohydride to ε-N-monomethyl-lysine (B). A second round of the reaction generates the final product ε-N,Ndimethyl-lysine (C).

tions (aqueous buffer, pH 7–8), the accessible lysine residues on proteins are completely converted to the ε-N,N-dimethyl derivatives. The reaction (Fig. 1) occurs in two distinct phases. Initially, the ε-amino group of the lysine forms an adduct with the formaldehyde to produce a Schiff base. This then undergoes reduction by sodium cyanoborohydride to the monomethylamine derivative in the second part of the reaction. A further round of the reaction, which occurs more rapidly than the first produces the dimethyl derivative. The attractiveness of this modification is that dimethylation of the lysine side chain is a relatively small chemical change. The pKa is only slightly affected and may even remain unchanged (5). Because of this, the residue maintains its ionization properties and the potential for the formation of the same ion-pair interactions as in the unmodified protein remains, although there is some loss of hydrogen-bonding capacity. Nevertheless, the modification is a mild one and unlikely to significantly perturb the native enzyme structure. A major use of any chemical modification procedure is the incorporation of isotopic labels at specific positions in proteins. Reductive methylation experiments that incorporate 13C have been used to probe the environment of lysine side chains in proteins using nuclear magnetic resonance (NMR) spectroscopy (7). The incorporation of radiolabels into proteins by reductive methylation enables the number of lysines that are accessible to be determined. In the case of DNA-binding proteins, the labeling is carried out on the free protein and the DNA–protein complex. This immediately provides information about the number of lysines present in the DNA-binding site. Subsequent peptide mapping strategies allow identification of specific residues, the degree of labeling at particular sites is then used to derive the location of that residue within the protein. In this way, [3H] formaldehyde has been used as a source of radiolabel to probe the role of the lysine residues of the core histones in the nucleosome


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(8), the interaction of the linker histone H5 with nucleosomes in long chromatin (9), and the role of lysine residues in a specific DNA–protein complex between the type I DNA methylase M.EcoR124I (10) and a short DNA duplex containing its recognition site. Subheading 3. contains a set of protocols to enable quantification of the number of lysine residues susceptible to reductive methylation in a protein and a DNA–protein complex. The peptide mapping and N-terminal proteinsequencing procedures required to identify and determine the extent of modification at individual residues are also described. Because the modification reaction is sensitive to contaminants in commercial grades of sodium cyanoborohydride (most likely cyanide), a method to recrystallize the reagent (6) is described in Subheading 3.1. In order to obtain quantitative information about the total extent of protein modification and/or determine the relative accessibility of individual residues, it is vital that the specific activity of the [3H] formaldehyde be determined accurately. Subheading 3.2. describes a protocol to do this using a simple peptide substrate α-melanocyte-stimulating hormone (α-MSH). The data from this experiment then allow quantitative conclusions to be drawn from the subsequent protein protection and peptide mapping experiments that are described in Subheadings 3.3. and 3.4. 2. Materials 2.1. Reagents and Materials 1. Reagents were obtained as follows: urea, formic acid, hydrochloric acid and glacial acetic acid (ARISTAR grade), dichloromethane (dried, AnalaR), glycine, Tris (hydroxymethyl) methylamine, sodium chloride and Na 2 EDTA (AnalaR), trifluoroacetic acid and water (HiPerSolv grade), acetonitrile (far UV HiPerSolv grade), and sodium cyanoborohydride (Schuchardt), all from Merck; dithiothreitol (DTT) and HEPES (both molecular biology grade), α-MSH and silica gel (Type III, indicating) from Sigma [ 3H] formaldehyde (approx 100 Ci/mol). NEN radiochemicals; filter paper (No. 1) from Whatman; dialysis membrane (Slide-A-Lyser) from Pierce; scintillation fluid (Ecoscint™ H) from National Diagnostics); trypsin (sequencing grade) and Pefabloc™ from Boehringer Mannheim. 2. DNA-binding protein (approx 10 mg). 3. Stock solution of oligonucleotide duplex, 300 µM (1 µmol synthesis, high-performance liquid chromatography [HPLC] purified). 4. Formaldehyde (Sigma): 37% solution and 15% methanol stabilizer. 5. 8 M urea and 50 mM acetic acid. 6. 10 mM HEPES, pH 7.5. 7. 1 M Na-glycine, pH 7.0. 8. 50 mM TBS: 10 mM Tris-HCl, 50 mM NaCl, and 1 mM EDTA, pH 7.5. 9. 2 M Tris base.

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2.2. Equipment 1. HPLC: binary gradient formation using high-pressure-mixing, variable-wavelength ultraviolet (UV) absorbance detector and fraction collection are required. 2. Reverse-phase HPLC columns: semipreparative C3 (9.4 × 250 mm), analytical C3 (4.6 × 250 mm), and analytical C18 (4.6 × 250 mm). Zorbax 300SB or an equivalent wide-pore packing is recommended. 3. High-resolution anion-exchange column (e.g., TSK-GEL, DEAE-NPR [4.6 × 35 mm]). 4. Freeze-dryer. 5. Scanning (UV/visible) spectrophotometer. 6. Liquid scintillation counter. 7. Protein-sequencing facilities.

3. Methods 3.1. Recrystallization of Sodium Cyanoborohydride 1. Dissolve 44 g of sodium cyanoborohydride in 100 mL of acetonitrile and remove any undissolved material by centrifugation. 2. Add 600 mL of dichloromethane to the mixture place in a sealed container and allow the sodium cyanoborohydride to precipitate overnight at 4°C. 3. Filter the mixture (Whatman filter paper No. 1) to collect the precipitate and wash with an additional 100 mL of cold dichloromethane. 4. Allow the powder to dry and store in a vacuum desiccator containing silica gel. Prepare aqueous stock solutions immediately before use.

3.2. Determination of the Effective Specific Activity of 3H Formaldehyde There is a substantial variation in the effective specific activity of different batches of [3H] formaldehyde. In order to accommodate this, it is necessary to determine the effective specific activity of each individual batch. A convenient way to do this is to use a simple peptide substrate for which the number of accessible amino groups is known. The effective specific activity can then be calculated from the amount of radioactivity that can be incorporated into the fully modified peptide. For this purpose the peptide hormone α-MSH (Ac-SYSMEHFRWGKPV-NH2) is recommended (see Note 1). 1. Redissolve the contents of a 1-mg vial of α-MSH in 1 mL of 8 M urea and 50 mM acetic acid. 2. Equilibrate a C18 reverse-phase column in 2% acetonitrile and 0.05% (v/v) trifluoroacetic acid (TFA) at a flow rate of 1 mL/min and set the UV absorbance detector to 280 nm. Apply the peptide in approx 200-µg aliquots and elute with a 0–60% increasing gradient of acetonitrile, 0.05% TFA (v/v) over 60 min. 3. Collect the peptide-containing fractions from multiple runs, pool, and lyophilize them. Store the purified peptide at –20°C in a box containing silica gel.


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4. Dissolve 100 nmol of purified α-MSH in 0.5 mL of 10 mM HEPES, pH 7.5. Add 10 mM sodium cyanoborohydride (freshly made) followed by 2 mM [3 H] formaldehyde (see Notes 2 and 3) and incubate at 24°C for 3 h. After this time add a second aliquot of the reagents and allow the reaction to continue for an additional 3 h. 5. Adjust the sample to 8 M urea and 50 mM acetic acid and purify the modified peptide by reverse-phase chromatography using the same gradient as in step 2. Pool the fractions, lyophilize, and store at –20°C in a box containing silica gel. 6. To determine the specific activity of the modified peptide, redissolve the labeled peptide in 1 mL of water and determine its concentration from the absorbance at 280 nm (ε280 = 7000/M/cm). Add 10 µL to 1 mL of liquid scintillant (Ecoscint H or equivalent), mix well, and determine the amount of incorporated radioactivity by liquid scintillation counting. The specific activity of the labeled peptide (σα-MSH) (in nCi/nmol) is calculated from Eq. 1. The effective specific activity of the [3H] formaldehyde (σ[3H] formaldehyde) is simply half this value (Eq. 2) (see Note 4): σa-MSH = (dpm)/(No. nmoles counted × 2220)

(1)

σ[3H] formaldehyde = 1/2 σα-MSH

(2)

3.3. Surface Labeling of Protein and DNA–Protein Complex by Reductive Methylation and Quantification of Residues Modified Prior to mapping the positions of modified lysine residues, it is necessary to determine the number of residues susceptible to reductive methylation and the proportion of these that are protected by the presence of DNA. 1. Prepare approx 3.0 mL of a solution of the DNA-binding protein in 10 mM HEPES, 50 mM NaCl, and 1 mM EDTA (pH 7.5) by either dialysis or buffer exchange. The concentration of DNA-binding protein in this solution should be 1–2 mg/mL. 2. Take 1.5 mL of protein and add an equimolar amount, or slight excess, of the DNA duplex from a concentrated stock solution to form the DNA–protein complex. 3. Withdraw a 300-µg aliquot from the free protein and the DNA–protein complex samples and add glycine to 50 mM from a neutral 1 M stock solution. Dilute the samples to 1 mL with 50 mM TBS and load into dialysis cassettes (see Note 5). Dialyze against 2 L of 50 mM TBS at 4°C. These samples are controls for efficiency of the whole procedure and also serve as the “zero” time points (see below). 4. To the remaining solutions, add a 30-fold molar excess of sodium cyanoborohydride over the total lysine content of the protein. Then, add a 10-fold molar excess of [3H] formaldehyde (see Note 6) and incubate at 24°C. 5. At timed intervals (10 min up to 5 h) withdraw 300 µg samples of protein from each time-course. Quench the reaction by the addition of 50 mM glycine and

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dilute to 1 mL with 50 mMTBS. Load the samples into dialysis cassettes and dialyze overnight against 2 L of 50 mM TBS using at least 3 changes. 6. After extensive dialysis, remove all the samples from the cassettes. For freeprotein samples, continue from step 7 onward. Samples from the DNA–protein complex time-course need to be processed to remove the DNA as follows. Equilibrate a small-volume (1 mL or less) high-performance anion exchange (see Note 7) HPLC column (analytical TSK-GEL, DEAE-NPR [4.6 × 35 mm] or an equivalent) in 50 mM TBS at a flow rate of 1 mL/min. Set the UV detector to 280 nm. Apply each sample from the time-course to the column and collect any flowthrough. Elute the protein (if bound) and the DNA by application of an increasing NaCl gradient from 0.05 M to 1.0 M over 50 column volumes. Collect the protein-containing fractions and proceed. 7. Determine the molar concentration of the samples from each time-course (including the “zero”) using the absorbance at 280 nm. Add 10 µL of each sample to 1 mL of liquid scintillant, mix well, then determine the amount of incorporated radioactivity at each time-point by liquid scintillation counting. 8. Calculate the specific activity (in nCi/nmol) of the labeled protein (σprotein) at each time-point of the reaction using Eq. 3. Then, using the value for the effective specific activity of the [3H] formaldehyde determined in Subheading 3.2. calculate the number of lysine residues modified at each point in the time-course using Eq. 4 (see Note 8): σprotein = (dpm)/(No. nmoles counted × 2220)

(3)

No. modified lysines = σ(protein) = 2σ([3H] formaldehyde)

(4)

9. Plot the number of lysine residues modified against time and fit the data to a single exponential process using Eq. 5 (Fig. 2). In most cases, the data should fit well to this model (see Note 9) and the total number of modifiable lysine residues is then given by the limit value (L). Fitting the data in this manner also allows a rate constant (k) for the incorporation of radiolabel to be derived. Both of these parameters can be affected by DNA binding. No. modified lysines = L(1–e–kt)

(5)

10. Compare the fitted curves of the time-course for the reaction of free protein and for the DNA–protein complex (Fig. 3). Formation of the DNA–protein complex may well reduce L, indicating the presence of a population of strongly protected lysines. At the same time, differences in k are likely to arise from an overall lowering of the rate of modification because of decreased accessibility of lysine residues in the presence of DNA.

3.4. Pulse Chase Labeling of Proteins If the fraction of lysine residues protected by DNA is large, as determined in Subheading 3.3., then a pulse-labeling procedure carried out on the free protein will reveal which lysine residues are surface accessible and likely to be


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Fig. 2. Time-course of reductive methylation of the type I DNA methyltransferase M.EcoR124I. The curve is the best fit of the data to a single exponential (L = 41, k = 0.019/min).

Fig. 3. The effect of DNA binding on reductive methylation of the DNA recognition subunit (HsdS) from M.EcoR124I. The upper curve is a best fit to the data from a time-course for the modification reaction of free protein (L = 19, k = 0.019/min). The lower curve is the best fit to the data from a time-course for modification of the protein in the DNA–protein complex (L = 14, k = 0.004/min).

involved in DNA binding. If only a small number of lysine residues are protected by DNA, then a modification to the procedure should be undertaken (see Note 10).

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1. Prepare 1 mL of DNA-binding protein at a concentration of 1–2 mg/mL in 50 mM TBS by either dialysis or buffer exchange. 2. Add sodium cyanoborohydride to a 30-fold molar excess over the total lysine content of the protein and then initiate the chemical modification reaction by the addition of [3 H] formaldehyde at a 10-fold molar excess. Incubate at 24°C, the length of time will depend on the results from the experiments in Subheading 3.3. Aim to modify for a length of time when the reaction is about 50% complete (see Note 11). This will probably be between 10 and 60 min. 3. At the end of the pulse, quench the reaction by the addition of 50 mM glycine and dialyze overnight against 1 L of 50 mM TBS. Change the dialysis buffer at least three times. 4. Equilibrate a semipreparative C3 reverse-phase column (9.4 × 250 mm) in 5% acetonitrile and 0.05% (v/v) TFA at a flow rate of 3 mL/min and set the UV detector to 225 nm. Remove the protein from the dialysis cassette, add urea to a final concentration of 8 M and DTT to 50 mM. Incubate the sample briefly at room temperature, acidify by the addition of 100 mM acetic acid, and apply the protein to the column. Elute with a 5–65% gradient of acetonitrile and 0.05% (v/v) TFA over 60 min. Collect the protein containing fractions and lyophilize them. 5. Redissolve the modified protein in 1 mL of 8 M urea and 10 mM HEPES (pH 7.5) and determine the protein concentration from the absorbance at 280 nm. At this point, the extent of label incorporation should be determined as in Subheading 3.3., steps 7 and 8. 6. To “chase” the reaction with unlabeled reagent, add a 30-fold molar excess of sodium cyanoborohydride over the total lysine content followed by a 10-fold excess of unlabeled formaldehyde. Incubate for 3 h at 24°C, then add a second aliquot of these reagents and continue the reaction for a further 3 h. 7. Add DTT to a final concentration of 50 mM, incubate briefly at room temperature then acidify with 100 mM acetic acid. Purify the fully modified protein by reverse-phase chromatography as in step 4. Lyophilize the fractions containing protein and store in aliquots of approx 2 nmol at –20°C in a box containing silica gel. 8. Redissolve a 2 nmol aliquot of modified protein in 100 µL of 0.9% formic acid (see Note 12). Ensure that the sample is fully dissolved then dilute to 500 µL with dH2O and titrate to pH 8 by the addition of 35 µL of 2 M Tris base. 9. Dissolve the contents of a vial of sequencing grade trypsin (see Note 13) in 1 mM HCl to give a concentration of 1 mg/mL. Add the trypsin to the protein to give an enzyme to a substrate ratio of 1:10 (w/w) and incubate at 37°C for approx 18 h. To increase the efficiency of cleavage add the trypsin in three aliquots at roughly 4-h intervals. Terminate the digest by the addition of 1 mM Pefabloc and store at –20°C until required. 10. Equilibrate an analytical C3 reverse-phase HPLC column (4.6 × 250 mm) in 2% acetonitrile and 0.05% (v/v) TFA at a flow rate of 1 mL/min and set the UV absorbance detector to 214 nm. Adjust the tryptic digests to 8 M urea and 50 mM DTT and incubate briefly at room temperature. Acidify the mixture by the addition of 100 mM acetic acid and apply to the column. Elute the peptides with an increasing gradient of acetonitrile collecting 250 µL fractions. For a complex


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Fig. 4. Separation of reductively methylated peptides from pulse-labeled HsdS by C4 reverse-phase chromatography. The absorbance at 214 nm and the amount of radioactivity (nCi) are plotted for each fraction.

11.

12.

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15.

mixture of peptides, the following gradient works well: 2–35% in 45 min followed by 35–60% in 20 min. It may be necessary to alter this for the particular protein under investigation. Remove 25 µL from each fraction, add 1 mL of liquid scintillant, and determine the level of radioactivity by liquid scintillation counting. Pool the fractions across each peak and lyophilize them. Store at –20°C in a box containing silica gel until required. Construct an overlaid chromatogram as in Fig. 4 and use this to select peaks with an apparently high specific activity (see Note 14). These peaks require further fractionation by C18 reverse-phase chromatography (see Note 15) before ultimately submitting them for N-terminal amino acid sequencing. Equilibrate an analytical C18 reverse-phase column in 2% acetonitrile and 0.05% TFA at a flow rate of 1 mL/min and set the UV detector to 214 nm. Redissolve each selected peak in 500 µL of 8 M urea and 100 mM acetic acid and apply to the column. Elute the bound peptides with a linear gradient of acetonitrile (2–50% in 70 min) and collect 250-µL fractions. Remove 25 µL from fractions across each peak and determine the incorporated radioactivity as in step 11. Lyophilize the remainder and store at –20°C in a box containing silica gel until required. Analyze each purified peptide using automated N-terminal amino acid sequencing. The objective is to determine the number of pmoles of each amino acid released at each cycle of the sequencing reaction and also to determine the amount of radioactivity associated with the residue. A suggested method for doing this is described in Note 16.

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3.5. Data Analysis The data from the N-terminal sequencing are used to determine the specific activity of each modified lysine as follows. Plot the number of pmoles of each residue released at each cycle pmole(n) versus the cycle number (n); then, fit these data to Eq. 5 (see Fig. 5A). E is the efficiency of the sequencing process (usually around 90%) and pmole(0) is the amount of starting material. pmole(n) = pmole(0) × En

(5)

As PTH-dimethyl-lysine is not a standard amino acid, the number of pmoles of modified lysine is not determined directly from integration of the HPLC trace. Instead, the value can be calculated by interpolation of the fitted curve for the cycle at which the dimethyl-lysine was released. Plot the amount of radioactivity released at each cycle versus the cycle number (Fig. 5B). Significant quantities of radioactivity should only be present at a cycle where a modified lysine is present. Combine the data from the two plots and use Eq. 1 to calculate the specific activity (σlysx); then, determine the fractional modification (σlysx/σα-MSH) for each dimethyl-lysine in a peptide. The value of this ratio is proportional to the accessibility of the residue during the pulse part of the chemical modification reaction. Values close to unity indicate a high degree of accessibility, whereas values close to zero indicate a residue that is inaccessible to chemical modification. These data can be used to build up a picture of the protein surface and identify clusters of lysines that are potential surfaces for DNA binding. Residues identified by these methods are then targets for site-directed mutagenesis experiments. 4. Notes 1. The α-MSH peptide contains only a single lysine and has a blocked N-terminus. The presence of the two aromatic residues allow its concentration to be determined accurately from its UV absorbance at 280 nm (ε280 = 7000/M/cm). We have extensively characterized the reductive methylation reaction with this peptide using NMR and mass spectroscopy (10). Under the reaction conditions used in Subheading 3.2., >95% of the product is dimethylated and the remainder monomethylated. In principle, any peptide substrate with a known number of free amino groups and for which the concentration can be measured accurately could be used to determine the effective specific activity. However, if a different peptide substrate is used, it is advisable to extensively characterize the reaction in the same way. On a routine basis, if access can be gained to a mass spectrometer, it may be worthwhile checking the completeness of the reaction in this way. 2. [3H] Formaldehyde from NEN is supplied as a 0.3 M aqueous solution in snapoff glass vials. Make sure the whole contents of the vial are at the bottom and then leave on ice for 10 min before breaking the seal. After opening, transfer the


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Fig. 5. Identification of radiolabeled lysine residues in the tryptic peptide D190N220 from the reductively methylated pulse-labeled HsdS subunit from M.EcoR124I. (A) The pmole yield at each cycle of the Edman degradation sequencing reaction. Fitting of the data to Eq. 5 allows the yield of dimethyl-lysine to be determined by interpolation. (B) A histogram showing the amount of radioactivity released at each cycle of the same sequencing reaction. The combination of radioactivity and picomole released at each cycle allows estimation of the specific activity of individual residues. contents to a screw-cap microfuge tube and store at 4°C. If possible, use all of the reagent within 1–2 d of opening. 3. The efficiency of the reductive methylation of proteins is greatly reduced by the presence of amines in the solution because of competitive inhibition. Thus, com-

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Taylor and Webb monly used buffers such as Tris–HCl and triethanolamine have to be avoided. HEPES and phosphate are good alternatives. A further problem is the potential for the formaldehyde–lysine adducts to undergo side reactions leading to unwanted protein crosslinking. To prevent this, the sodium cyanoborohydride should be added to the protein solution prior to addition of the formaldehyde. The stoichiometry of the reductive methylation reaction dictates that two molecules of formaldehyde are required to complete the modification of a lysine residue to ε-N,N-dimethyl-lysine (see Fig. 1). Because of this, the effective specific activity of the [3H] formaldehyde is half the value determined for the fully dimethylated peptide. For dialysis of small volumes, (0.5–2 mL) Slide-A-Lyser cassettes (Pierce) are extremely useful. We find that a 1-mL sample volume is easy to inject and recover from the cassette without large losses of sample and without large dilution. If necessary, a smaller volume could be used. The amount and the exact ratio of the reagents used for the reaction are somewhat empirical. The major concern is the prevention of side reactions resulting from reactive formaldehyde–lysine adducts. For a detailed account, see ref. 6. Briefly, the concentration of formaldehyde needs to be at an excess over the number of lysine residues to drive the reaction to completion, but not so high as to favor protein crosslinking. The other requirement is that the sodium cyanoborohydride be in excess over the formaldehyde to ensure efficient reduction of the Schiff bases. To separate the DNA from protein, DEAE or Q ion-exchange columns are the method of choice. DNA oligonucleotides will bind very strongly to these matrices and the protein either can be recovered from the flowthrough or will elute earlier in a NaCl gradient. An alternative is to use heparin–Sepharose or, for basic proteins, a cation-exchange resin. If the chromatographic separation of the DNA from the protein is problematic, treat each sample with DNase I (FPLCpure, Pharmacia) before application to the column. The N-terminus of the protein can also be reductively methylated. If the protein is relatively small or the total number of modified residues is low, then it is worthwhile to consider this when calculating the extent of modification. If the time-course is extended to 5 h incubation, the reaction should be complete and the data will usually fit well to a single exponential process. Occasionally, this is not the case—for instance, if the protein contains several distinct populations of lysines with different kinetics. In this case, a more complex model will be needed to deconvolve the various classes of reacting species. The pulse-labeling method involves treating the protein with a short pulse of labeled formaldehyde followed by a “cold” chase. This will identify all the surface lysines and provide information about their accessibility. If a large proportion of the total number of modifiable lysine residues are protected by DNA, then a pulse chase procedure of this kind will identify residues likely to be involved in DNA binding. However, if only a small proportion of lysines are protected by DNA, then an initial “cold” labeling should be performed on the DNA–protein


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complex, followed by separation of the protein from the DNA before the pulse with labeled reagents is applied. This is a compromise between getting enough label into the protein to allow the sites of modification to be easily determined and providing for differential labeling at individual sites so that information about the accessibility of each lysine can be obtained. There may be some difficulty in redissolving the lyophilized protein in the aqueous buffers required for tryptic digestion (e.g., 20 mM Tris-HCl, pH 8.0). The formic acid strategy described works well in some cases but is not guaranteed. An alternative is to dissolve the sample in 100 mM Tris-HCl and 8 M urea pH 8.0 and then to add an equal volume of trypsin in 1 mM HCl such that the final urea concentration is 4 M and the trypsin concentration 1:10 (w/w). The objective of the proteolytic digest is to produce peptides of an optimal length (5–30 amino acids) for quantitative analysis by automated Edman degradation. A tryptic digest of a reductively methylated protein will produce an arginine specific digest, as ε-N,N-dimethyl-lysine residues are not substrates for tryptic cleavage (11). Such a digest will yield some peptides that are suitable for N-terminal sequencing but will probably not cover the entire protein. In order to produce further peptides of suitable length, other proteases and chemical cleavage reagents should be investigated. The usefulness of these agents can vary substantially. In general, the best enzymatic alternatives are chymotrypsin and V8 protease. Cyanogen bromide is the best alternative for chemical cleavage. The main criterion for selection of peaks is an apparent high specific activity, indicating that surface accessible lysines are present in peptides eluted within the peak. Additional information about the location of buried lysines within the protein can be gained by sequencing peptides which show very low or apparently no label incorporation. Although this could yield valuable data, one should be aware that some of these “cold” peaks are likely to be peptides derived from trypsin. After an initial separation of a complex mixture of reductively methylated peptides, by C3 reverse-phase chromatography, a further fractionation of peptides using either C8 or C18 is highly recommended. Often a peak with an apparently high specific activity taken from an initial C3 separation will resolve into multiple components on C8 or C18, only some of which are labeled. Avoid loading peptides eluted from a C3 column at high acetonitrile concentrations (>50%) onto columns with longer alkyl-chain-bonded phases as the interaction between the sample and the bonded phase may be too strong for efficient recovery. For these larger, more hydrophobic peptides, it may be better to reapply to a C3 column and then elute with a different gradient to the one used initially. Alternatively, redigest with a different enzyme and separate the products on a C18 column. A simple and effective way to quantify the degree of incorporated label at individual lysine residues involves splitting the peptide sample during the automated Edman degradation sequencing reaction; most sequenators are equipped with this facility. After extraction of each 2-analino–5-thiazolinone-derivatized amino

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Taylor and Webb acid, split the sample and divert 50% to a fraction collector. Convert the remainder to the phenylthiohydantoin derivative and identify the residue by on-line HPLC analysis in the usual way. Calculate the number of picomoles of the newly released residue by integration of the HPLC peak. Determine the amount of radioactivity associated with the residue by liquid scintillation counting of the material that was diverted to the fraction collector. This method may require modification, depending on the configuration of the available sequenator.

References 1. Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents for Protein Modification. CRC, Boca Raton, FL. 2. Hunter, M. J. and Ludwig, M. L. (1962) J. Am. Chem. Soc. 84, 3491–3497. 3. Means, G. E. and Feeney, R. E. (1968) Reductive alkylation of amino groups in proteins. Biochemistry 7, 2192–2201. 4. Means, G. E. and Feeney, R. E. (1995) Reductive alkylation of proteins. Anal. Biochem. 224, 1–16. 5. Jentoft, N. and Dearborn, D. G. (1983) Protein labelling by reductive alkylation. Methods Enzymol. 91, 570–579. 6. Jentoft, N. and Dearborn, D. G. (1979) Labelling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 254, 4359–4365. 7. Zhang, M., Thulin, E., and Vogel, H. J. (1994) Reductive methylation and pKa determination of the lysine side chains in calbindin D9k. J. Protein Chem. 13, 527–535. 8. Lambert, S. F. and Thomas, J. O. (1986) Lysine-containing DNA-binding regions on the surface of the histone octamer in the nucleosome core particle. Eur. J. Biochem. 160, 191–201. 9. Thomas, J. O. and Wilson, C. M. (1986) Selective radiolabelling and identification of a strong nucleosome binding site on the globular domain of histone H5. EMBO J. 5, 3531–3537. 10. Taylor, I. A., Webb, M., and Kneale, G. G. (1996) Surface labeling of the type I methyltransferase M.EcoR124I reveals lysine residues critical for DNA binding. J. Mol. Biol. 258, 62–73. 11. Poncz, L. and Dearborn, D. G. (1983) The resistance to tryptic hydrolysis of peptide bonds adjacent to N-epsilon, N-dimethyllysyl residues. J. Biol. Chem. 258, 1844–1850.

Limited Proteolysis of Protein–Nucleic Acid

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22 Limited Proteolysis of Protein–Nucleic Acid Complexes Simon E. Plyte and G. Geoff Kneale 1. Introduction Limited Proteolysis is a useful structural probe for investigating the globular nature of proteins by preferentially digesting the more accessible regions often found between domains. Generally, proteases require a small region of polypeptide chain possessing conformational flexibility for accommodation in the active site (1). The regions of a protein possessing conformational flexibility are often found between tightly folded domains and are, therefore, preferential sites for proteolysis. In practice, limited proteolysis is achieved by dilution of the enzyme sufficiently so that it will only digest the most accessible regions leaving the domains intact. Digestion of protein–nucleic acid is often advantageous in that the DNA may provide steric protection of the DNAbinding domain not afforded by the free protein. The generation of domains by limited proteolysis relies directly on the tertiary structure of the protein under investigation and provides much firmer evidence for their existence than that provided by sequence homology. An increasing number of nucleic-acid-binding proteins are known in which regions of their polypeptide chain are folded separately into compact globular domains, each possessing a distinctive function. For example, digestion of the A1 heterogeneous nuclear ribonucleoprotein (A1 hnRNP) with Staphylococcus aureus V8 protease produces two discrete domains, both capable of binding single-stranded nucleic acids (2,3). Similarly, digestion of the Pf1 gene 5 nucleoprotein complex results in the production of a 12-kDa domain that retains much of the single-stranded DNA-binding ability of the intact protein (4). Further, using limited proteolysis, a cryptic DNA-binding domain was revealed in the COOH terminus of yeast TFIIIB70 and a core ssDNA-binding domain was From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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generated, possessing increased binding affinity, in human replication protein A (5,6). In addition to its use for the analysis of domain structure, limited proteolytic fragments from Escherichia coli DNA gyrase B, for example, permitted the successful crystallization and structure determination of one of its domains (7).

1.1. Strategy The strategy adopted for the limited proteolysis of nucleoprotein complexes can be considered in four parts: optimization of the proteolysis, characterization of the proteolysed complex, purification of the DNA-binding domains, and sequence characterization of the fragment(s).

1.1.1. Proteolysis of Nucleoprotein Complex The nucleoprotein complex should be digested with various proteases to establish which conditions are optimal for generating a protease-resistant domain. We routinely vary two parameters (enzyme/substrate ratio and time of digestion) when determining the best conditions for limited proteolysis. However, other parameters, such as temperature, ionic strength, and pH may also be varied. To determine the appropriate enzyme/substrate ratio for a particular protease, the nucleoprotein complex is digested at several enzyme/substrate ratios, removing samples at regular time intervals for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The appearance of a discrete domain, resistant to further degradation (even if only transiently), is evidence for the existence of a domain, although not necessarily one that binds DNA. Choice of protease is often critical (see Table 1). Initially, it is best to try a relatively nonspecific enzyme (e.g., papain) because this decreases the likelihood of activity being dependent on primary sequence rather than tertiary structure.

1.1.2. Preliminary Characterization of DNA-Binding Properties of the Proteolyzed Nucleoprotein Complex An initial indication of DNA binding can be found during the proteolysis experiment by removing two aliquots for gel analysis that can be run on polyacrylamide or agarose gels appropriate for the size of complex in the presence and absence of the denaturant SDS. A retardation in the mobility of the DNA (seen under ultraviolet [UV] light) in the absence of SDS implies that the fragment is still associated with DNA and constitutes a DNA-binding domain. However, this does not prove that the proteolyzed fragment is a discrete DNAbinding domain; it is possible that the nucleoprotein complex has only been “nicked” by the protease and maintains its native tertiary structure by noncovalent interactions. Therefore, it is necessary to purify the domain and fully characterize its DNA-binding properties.


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Table 1 Useful Enzymes for Limited Proteolysis Enzyme

Substrate specificity

α-Chymotrypsin

Preferentially cuts C-terminally to aromatic amino acids Elastase Cuts C-terminally to aliphatic noncharged amino acids (e.g., Ala, Val, Leu, Ile, Gly, Ser) Endoproteinase Arg-C Cuts C-terminally to arginine residues Endoproteinase Lys-C Cuts C-terminally to lysine residues Papain Nonspecific protease but shows some preference for bonds involving Arg, Lys Gln, His, Gly, and Tyr Pepsin Nonspecific protease Subtilisin Nonspecific protease Trypsin Cuts C-terminally to lysine and arginine residues Endoproteinase Glu-C Cuts C-terminally to glutamic acid (V8 protease) and or aspartic acid residuesa

Inhibitors Aprotinin, PMSF, DFP, TPCK, cymostatin PMSF, DFP

DFP, TLCK Aprotinin, DFP PMSF, TPCK, TLCK, leupeptin, heavy metal ions Pepstatin DFP, PMSF DFP, PMSF, TLCK DFP

Abbreviations used: DFP, diisopropyl fluorophosphate (extremely toxic!); PMSF, phenylmethyl sufonyl fluoride; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; TLCK, Nα-ptosyl-l-lysine chloromethly ketone. aWill cut C-terminally to glutamic acid residues in ammonium bicarbonate, pH 8.0, or ammonium acetate, pH 4.0; will cut C-terminally to glutamic and aspartic acid residues in phosphate buffer, pH 7.8.

1.1.3. Purification of the DNA-Binding Domain Purification of the fragment can make use of the fact that it will still be associated with DNA. Ultracentrifugation of the proteolyzed nucleoprotein complex (if large fragments of DNA are used) concentrates the domain and removes residual protease and small proteolytic fragments. The proteolyzed nucleoprotein complex can then be dissociated and the domain further purified if necessary. Alternatively, the DNA-binding fragment can be purified by affinity chromatography on DNA agarose. Several techniques are available to determine whether the purified domain binds DNA (discussed in several chapters) and include gel retardation assay, a variety of footprinting techniques, fluorescence spectroscopy, and circular dichroism.

1.1.4. Determination of the Amino Acid Sequence of the Domain N-Terminal sequencing and amino acid analysis of the purified DNA-binding domain should be sufficient to establish the sequence of the domain, if the

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native amino acid sequence is known. Alternatively, N-terminal sequencing and mass spectroscopy should enable unambiguous identification of the domain. If certain proteases have been used to generate the domain (e.g., trypsin, α-chymotrypsin, endoproteinase Arg-C, and so forth), the C-terminal amino acid may also be known. If there are still ambiguities, carboxypeptidase digestion of the fragment can also be used to help identify the C-terminal residues, although this is not always reliable. If this still does not yield an unambiguous result, one must resort to amino acid sequencing of the entire fragment. 2. Materials 1. Spectra-Por dialysis membrane washed thoroughly in double-distilled water. 2. All proteases should be of the highest grade available and treated for contaminating protease activity, if necessary. A list of useful enzymes and their inhibitors is given in Table 1. 3. Buffers should be AnalR grade or higher and made up in double-distilled water. 4. SDS–polyacrylamide gel stock solutions: Solution A: 152 g acrylamide and 4 g bis-acrylamide. Make up to 500 mL. Solution B: 2 g SDS and 30 g Tris base, pH 8.8. Make up to 500 mL. Solution C: 2 g SDS, 30 g Tris base, pH 6.8. Make up to 500 mL. When making up these solutions, they should all be degassed and filtered using a Buchner filter funnel. They should be stored in lightproof bottles and will keep for many months. 6. 10% ammonium persulfate (APS): dissolve 0.1 mg in 1 mL of dH2O. 7. 15% SDS–polyacrylamide gel: Mix together 8.0 mL of solution A, 4.0 mL of solution B, and 3.9 mL of dH2O. Add 150 µL of 10% APS and 20 µL of N,N,N',N'tetramethylethylene diamine (TEMED). Mix well and then pour between the plates. Immediately place a layer of dH20 (or butanol) on top of the gel to create a smooth interface with the stacking gel. When the resolving gel has set, pour off the water and prepare the stacking gel. This is done by adding 750 µL of solution A and 1.25 mL of solution C to 3.0 mL of dH2 O. Finally, add 40 µL of APS and 10 µL of TEMED, pour on the stacking gel, and insert the comb. Remove the comb as soon as the gel has set to avoid the gel sticking to the comb. 8. 10X SDS running buffer: 10 g SDS, 33.4 g Tris base, and 144 g glycine made up to 1 L. 9. High-methanol protein stain: technical-grade methanol 500 mL, 100 mL glacial acetic acid and 0.3 g PAGE 83 stain (Coomassie blue), made up to 1 L. 10. Destain solution: 100 mL methanol and 100 mL glacial acetic acid, made up to 1 L. 11. 2X SDS-PAGE loading buffer: 4% (w/v) SDS, 60 mM Tris-HCl, pH 6.8, 20% glycerol, 0.04% (w/v) bromophenol blue, and 1% (v/v) β-mercaptoethanol. 12. 6X agarose gel loading buffer: 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol, and 30% glycerol. 13. 6X agarose gel loading buffer plus SDS: as in item 12 plus 12% SDS (w/v). 14. TE buffer: 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA. 15. 5 M NaCl or MgCl2 (or other concentrated salt solutions for dissociation of the nucleoprotein complex [e.g., NaSCN]).


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3. Methods The method given here covers the first three objectives outlined in Subheading 1.1. Experimental details for the determination of the amino acid sequence of the fragment can be found in any standard text on protein chemistry. The following protocol was used for the generation of an 11-kDa DNA-binding domain from the Pf1 gene 5 protein (8). This protein binds cooperatively to ssDNA to produce a nucleoprotein complex of several million Daltons. Different nucleoprotein complexes will require different conditions of digestion and purification, but the basic principles remain the same.

3.1. Limited Proteolysis 1. Dialyze the nucleoprotein complex into the appropriate digestion buffer (see the manufacturer’s recommendations for the buffer, temperature of reaction, and inhibitor). We routinely digest the nucleoprotein complex at approx 1 mg/mL, but the concentration is not too critical. 2. Prepare 40 tubes containing 5 mL of 2X SDS loading buffer plus 1 µL of the appropriate protease inhibitor. Leave on ice. 3. Pipet 55 µL of the nucleoprotein complex (55 mg) into each of four tubes labeled 1:100, 1:200, 1:500, 1:1000, respectively. Place on ice until needed. 4. Dissolve the protease in digestion buffer to a concentration that will give an enzyme/substrate ratio of 1:100 (w/w) when 1 µL of the protease is added to 50 µL of nucleoprotein complex (i.e., 0.5 mg/mL). 5. Prepare three dilutions of the protease. In this case, the protease is diluted 1:2, 1:5, and 1:10 with digestion buffer that will result in a final enzyme substrate ratio of 1:200, 1:500, and 1:1000 (w/w). 6. Remove 5 µL of the nucleoprotein complex from each of the four tubes and add to 1 of the 40 tubes containing 2X loading buffer (plus inhibitor) and place on ice. This is the time = 0 tube and should be labeled accordingly. 7. Add 1 µL of the protease to the appropriate nucleoprotein solution (e.g., protease diluted 1:5 to the nucleoprotein solution marked 1:500) and incubate at the specified temperature. 8. Remove 5 µL samples every 15 min and add to 2X loading buffer (in the appropriately marked tube) and then place on ice. 9. At the end of the experiment, boil the samples and run an SDS polyacrylamide gel. The presence of a degraded fragment(s), resistant to further proteolysis, is evidence for a discrete domain (see Note 1). 10. Adjustment of the enzyme/substrate ratios, time course, and choice of enzymes is often necessary. The optimum conditions must be found by trial and error.

3.2. Purification of the DNA-Binding Domain 1. Digest a large quantity (several milligrams) of the nucleoprotein complex under the optimized conditions determined in Subheading 3.1. to produce the DNAbinding domain (see Note 2). Add the appropriate inhibitor and run a sample on SDS-PAGE to check the digestion.

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2. For very large nucleoprotein complexes, the proteolyzed complex can be purified away from the protease and small proteolytic fragments by ultracentrifugation. Spin the nucleoprotein complex at 229,000g (Beckman 70.1 Ti rotor) for 3 h at 4°C (see Note 3). Carefully wash the centrifuge tube with 4 mL of TE buffer, discard the washings, and resuspend the nucleoprotein complex in 2 mL of TE buffer on ice. Another ultracentrifugation step can be performed to remove all traces of the protease. For smaller nucleoprotein complexes, the DNA can be immobilized on a large resin (e.g., DNA cellulose) prior to interaction with the DNA-binding protein. Low-speed centrifugation can then be used to purify the DNA-associated domain. Sometimes, limited proteolysis can generate several fragments that bind DNA. These may arise from the same region of the protein; if so, this can be overcome by allowing the proteolysis to proceed further or by increasing the amount of protease. 3. Dissociate the proteolyzed nucleoprotein complex by the addition of salt to the appropriate concentration (see Note 4). The DNA can then be removed by ultracentrifugation (if sufficiently large) or nuclease digestion. If the DNA was originally bound on a solid support, then it can be removed by low-speed centrifugation (see Note 5). 4. Remove the high-salt buffer by desalting or dialysis. If the sample contains several different domains or a residual undigested protein, it will be necessary to purify the domains to homogeneity. Various chromatographic techniques are available to further purify the domains, including chromatofocusing, ion exchange, affinity, and gel filtration chromatography. These techniques permit recovery of the domain in a native state for further biochemical analysis. Alternatively, if the fragment is only to be used for sequence analysis, the mixture can be applied to a C3 reverse-phase high-performance liquid chromatography column and separated in an acetonitrile gradient. 5. If the sequence of the native protein is known, then the sequence of the DNAbinding domain can be established by N-terminal sequencing and amino acid analysis. Additionally, the mass of the fragment (determined by mass spectroscopy) should help locate the sequence of the DNA-binding domain.

4. Notes 1. Often during the experiment, a protease-resistant fragment is only transiently formed during complete digestion of the protein. If this occurs, vary some of the parameters (enzyme dilution, temperature, etc.) to try and prolong the lifetime of the fragment. 2. Scaling up of the digestion is not generally a problem and we routinely digest several milligrams (>10) of nucleoprotein complex if necessary. 3. The speed and duration of centrifugation will vary depending on the size of the nucleoprotein complex. For smaller complexes, ultracentrifugation may not be appropriate. 4. In many cases, a NaCl concentration between 1 M and 2 M is sufficient to dissociate the nucleoprotein complex. However, some nucleoprotein complexes


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remain associated above 2 M NaCl and require 1 M MgCl2 or 1 M NaSCN for dissociation (8). The appropriate salt concentration can be determined by SDSPAGE analysis of the pellet and supernatant after ultracentrifugation at different ionic strengths. 5. The DNA can also be removed by DNase digestion followed by gel fitration (i.e., desalting column) or by extensive dialysis against TE buffer.

References 1. Vita, C., Dalzoppo, D., and Fontana, A (1987) Limited proteolysis of globular proteins: molecular aspects deduced from studies on thermolysin, in Macromolecular Biorecognition (Chaiken, I., Chaiancone, E., Fontana, A., and Veri, P., eds.), Humana Press, Clifton, NJ. 2. Merril, B., Stone, K., Cobianchi, F., Wilson, S., and Williams, K. (1988) Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid binding pocket in the heterogeneous nuclear ribonucleoprotein. J. Biol. Chem. 263, 3307–3313. 3. Bandziulis, R., Swanson, M., and Dreyfuss, G. (1989) RNA binding proteins as developmental regulators. Genes Dev. 4, 431–437. 4. Plyte, S.E. and Kneale, G.G. (1993) Characterization of the DNA-binding domain of the Pf1 gene 5 protein. Biochemistry 32, 3623–3628 5. Huet, J., Conesa, C., Carles, C., and Sentenac, A. (1997) A cryptic DNA-binding domain at th COOH terminus of TFIIIB70 affects formation, stability and function of perinitiation complexes. J. Biol. Chem. 272, 18,341–18,349. 6. Bochareva, E., Frappier, L., Edwards, A., and Bochareva, A. (1998) The RPA32 subunit of human replication protein A contains a single stranded DNA-binding domain. J. Biol. Chem., 273, 3932–3936. 7. Wigley, D., Davies, G., Dodson, E., Maxwell, A., and Dodson, G. (1991) Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature 351, 624–629. 8. Kneale, G.G. (1983) Dissociation of the Pf1 nucleoprotein assembly complex and characterization of the DNA-binding protein. Biochem. Biophys. Acta 739, 216–224.

DNA–Protein Photocrosslinking

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23 Ultraviolet Crosslinking of DNA–Protein Complexes via 8-Azidoadenine Rainer Meffert, Klaus Dose, Gabriele Rathgeber, and Hans-Jochen Schäfer 1. Introduction In biological systems, photoreactive derivatives have been widely applied to study specific interactions of receptor molecules with their ligands by photoaffinity labeling (1–3). While the receptors are generally proteins (e.g., enzymes, immunoglobulins, or hormone receptors), the ligands differ widely in their molecular structure (e.g., sugars, amino acids, nucleotides, or oligomers of these compounds). The advantage of photoaffinity labeling compared with affinity labeling, or chemical modification with group-specific reagents is that photoactivatable nonreactive precursors can be activated at will by irradiation (Fig. 1). These reagents do not bind covalently to the protein unless activated. On irradiation of the precursors, highly reactive intermediates are formed that react indiscriminately with all surrounding groups. Therefore, after activation, a photoaffinity label, interacting at the specific binding site, can label all the different amino acid residues of the binding area. Today, aromatic azido compounds are mostly used as photoactivatable ligand analogs. They form highly reactive nitrenes upon irradiation because of the electron sextet in the outer electron shell of these intermediates (Fig. 2). In addition to the azido derivatives, photoreactive precursors forming radicals or carbenes on irradiation can be used as photoaffinity labels. All of these intermediates (nitrenes, e.g.) vigorously try to complete an electron octet (Fig. 3). To produce covalent crosslinks between proteins and DNA, various methods have been applied (4–11): ultraviolet (UV) irradiation, γ-irradiation, chemiFrom: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Fig. 1. Photoaffinity labeling of receptor proteins (e.g., enzymes) by photoactivatable ligand analogs (e.g., substrate analog/product analog). In the dark (upper line), the biological interactions of the protein with the ligand analog can be studied. On irradiation (lower line), the protein (enzyme) is labeled and inactivated by the substrate analog/product analog.

Fig. 2. Highly reactive photogenerated intermediates: radical (A), carbene (B), and nitrene (C).

cal methods, and even vacuum or extreme dryness. Besides these methods, photoaffinity labeling and photoaffinity crosslinking are helpful tools for the study of specific interactions between proteins and deoxyribonucleic acids. To date, many successful attempts have been made to photocrosslink proteins to nucleic acids using different photoactivatable deoxynucleotides. 5-bromo-, 5-iodo-, 5-azido-, and 5-[N-(p-azidobenzoyl)-3-aminoallyl]-2'-deoxyuridine5'-monophosphate (12–18), 4-thio-2'-deoxythymidine-5'-monophosphate (19), and 8-azido-2'-deoxyadenosine-5'-monophosphate (20,21) have been incorporated into deoxyribonucleic acids to bind DNA covalently to adjacent proteins (for a review see ref. 22). Here, we describe the synthesis of 8-azido-dATP (8-N3dATP), its incorporation into DNA by nick translation, and the procedure to photocrosslink azidomodified DNA to proteins (20,21).


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Fig. 3. Reactions of nitrenes. Cycloaddition to multiple bonds forming three-membered cyclic imines (1), addition to nucleophiles (2), direct insertion into C-H bonds yielding secondary amines (3), and hydrogen atom abstraction followed by coupling of the formed radicals to a secondary amine (4a, 4b).

2. Materials 2.1. Synthesis of 8-N3dATP 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

dATP (disodium salt, Boehringer Mannheim, Mannheim, Germany). Potassium acetate buffer: 1 M, pH 3.9. Bromine. Sodium disulfite (Na 2S2O5). Ethanol. DEAE–Sephadex A-25. Triethylammonium bicarbonate buffer: 0.7 M, pH 7.3. Dimethylformamide. Hydrazoic acid: 1 M in benzene. Triethylamine.

2.2. Characterization of 8-N3dATP 1. 2. 3. 4.

Silica gel plates F254 (Merck, Darmstadt, Germany). Cellulose plates F (Merck). Isobutyric acid/water/ammonia (66:33:1 v/v). n-Butanol/water/acetic acid (5:3:2 v/v).

2.3. Preparation of Azido-Modified DNA 1. DNA (e.g., pBR 322 or pWH 106). 2. Deoxyribonucleotides (dATP, dGTP, dCTP, dTTP, [α-32P]-dCTP).

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Fig. 4. Synthesis of 8-N3dATP. 3. DNase I (Escherichia coli, 2000 U/mg, Boehringer Mannheim) in 0.15 M NaCl and 50% glycerol. 4. 50 mM Tris-HCl, pH 7.2. 5. Magnesium sulfate (MgSO4). 6. Bovine serum albumin. 7. DNA polymerase I (E. coli, Boehringer Mannheim, No. 104493, purchased containing definite amounts of DNase I). 8. Ethylenediaminetetraacetic acid disodium salt (EDTA). 9. Sephadex A-25.

2.4. Photocrosslinking An ultraviolet lamp (e.g., Mineralight handlamp UVSL 25 at position “long wave”) emitting UV light at wavelengths of 300 nm and longer. 3. Methods 3.1. Synthesis of 8-N3dATP The synthesis of 8-N3dATP (Fig. 4) is performed principally by analogy to the synthesis of 8-N3ATP (23) (see Note 1). In the first step, bromine exchanges the hydrogen at position 8 of the adenine ring. Then, the bromine is substituted by the azido group. 1. Dissolve 0.2 mmol (117.8 mg) of dATP in 1.6 mL of potassium acetate buffer (1 M, pH 3.9) and add 0.29 mmol (15 µL) of bromine. Keep the reaction mixture in the dark at room temperature for 6 h (the absorption maximum shifts from 256 nm to 262 nm; see Note 2). 2. Reduce excessive bromine by addition of traces of (approx 5 mg) Na2S2O5 until the reaction mixture looks colorless or pale yellow. Pour the reaction mixture into 20 mL of cold ethanol (–20°C) and allow to stand for at least 30 min at –20°C in the dark. 3. Collect the precipitated deoxynucleotide by centrifugation and redissolve the residue in 0.5 mL of double-distilled water. Further purification is achieved by ionexchange chromatography over DEAE–Sephadex A-25 column (50 × 2 cm) with a linear gradient of 1000 mL each of water and triethylammonium bicarbonate (0.7 M, pH 7.3).


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Fig. 5. Elution profile (anion-exchange chromatography on DEAE–Sephadex A 25; elution buffer: linear gradient of 1000 mL each of water and 0.7 M triethylammonium bicarbonate (pH 7.3) of the reaction products of 8-N3 dATP synthesis: front (a), 8-N3dAMP (b), 8-BrdAMP (c), 8-N3dADP (d), 8-BrdADP (e), 8-N3dATP (f), 8-BrdATP (g), and probably a higher phosphorylated 8-azidoadenosine derivative (h). 4. Combine the fractions containing 8-bromo-dATP (8-BrdATP) (main peak of the elution profile) and dry by lyophilization. 8-BrdATP is obtained as the triethylammonium salt. The expected yield should be 65% (spectroscopically). 5. Dissolve 0.1 mmol (87.3 mg) of dried 8-BrdATP (triethylammonium salt) in 3 mL of freshly distilled dimethylformamide (see Notes 3 and 4). Add a dried solution of 0.8 mmol (34.4 mg) of hydrazoic acid (HN 3 ) in 800 µL of benzene and 0.8 mmol (111.3 µL) of freshly distilled triethylamine. Keep the reaction mixture in the dark at 75°C for 7 h (the absorption maximum shifts from 262 nm to 280 nm). 6. Evaporate the solvents under vacuum and redissolve the residue in 1 mL of water. Further purification is achieved by ion-exchange chromatography over DEAE– Sephadex A-25 as described in step 3. Figure 5 shows the elution profile of the chromatography (see Notes 4 and 5). 7. Combine the fractions containing 8-N3dATP and dry the solution by lyophilization. 8-N3dATP is obtained as the triethylammonium salt. Yield: 30% (spectroscopically). 8-N3dATP can be stored at –20°C in the dark freeze-dried (see Notes 6–8) or frozen in aqueous solution, pH 7.0.

3.2. Characterization of 8-N3dATP 1. Thin-layer chromatography (TLC). TLC is carried out on silica gel plates F254 or cellulose plates F. The development is performed in either isobutyric acid/water/ ammonia (66:33:1 v/v) or n-butanol/water/acetic acid (5:3:2 v/v).

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2. Ultraviolet absorbance. Record the UV absorbance spectrum of 8-N3dATP. It shows a maximum at 280 nm. The UV absorbance of 8-N3dATP is pH dependent (see Note 9). 3. Photoreactivity. The photoreactivity of 8-N3dATP is tested by two different methods. It can either be demonstrated by the spectroscopic observation of the photolysis (Fig. 6; see Note 10) or by the ability of the photolabel to bind irreversibly to cellulose on thin-layer plates on UV irradiation (Mineralight handlamp UVSL 25) prior to the development of the chromatogram. After development, most of the irradiated label is detected at the origin of the chromatogram in contrast to the nonirradiated control, which has completely migrated.

3.3. Preparation of Azido-Modified DNA Azido-modified and [32P]-labeled DNA are prepared by nick translation. For this purpose, the detailed and exact composition of the reaction medium depends strongly on the size as well as on the amount of the DNA to be modified. The optimal ratio of DNA, DNase I, and DNA polymerase I (Kornberg enzyme) should be tested in preliminary experiments (see Notes 11 and 12). Here, we describe the well-tested reaction conditions for the modification of plasmid pBR322 (4363 bp). The preparation of azido-modified pWH106 (4970 bp) can be performed analogously. 1. Add 17.3 pmol of pBR322 to a mixture of 50 nmol of dGTP, 50 nmol of dTTP, 50 nmol of 8-N3dATP, and 500 pmol of dCTP; prepare on ice. 2. Add 370 kBq of [α-32P]-dCTP (110 TBq/mmol) and 20 pg of DNase I (freshly prepared out of a stock solution of 1 mg of DNase I in 1 mL of 0.15 M NaCl and 50% glycerol). 3. Adjust the reaction medium to an end concentration of 50 mM Tris-HCl, pH 7.2, 10 mM MgSO4, and 50 mg/mL of bovine serum albumin (standard reaction volume: 100 µL). 4. Start the nick translation reaction by adding 100 U of DNA polymerase I from E. coli. 5. Incubate for 1 h at 15°C in the dark. 6. Stop the reaction by adding EDTA (final concentration: 20 mM). 7. Separate the unincorporated deoxyribonucleotides from photoreactive [32P]-labeled pBR322 by gel filtration over Sephadex A-25 column using a 1-mL syringe. 8. Precipitate photoreactive pBR322 by adding two volumes of cold ethanol and redissolve the precipitate in double-distilled water. Store the aqueous solution at –20°C in the dark.

Control nonphotoreactive DNA can be prepared analogously replacing the 8-N3dATP by 50 nmol of dATP.

3.4. Photocrosslinking 1. Prepare 20–30 µL aqueous solutions containing the photoreactive DNA (0.5 pmol) and the protein (1–25 pmol) to be cross-linked (see Notes 13 and 14).


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Fig. 6. Change of the optical absorption spectrum of 8-N3dATP on UV irradiation in Tris-HCl buffer (0.01 M, pH 7.0, 20°C). The irradiation time between two subsequent absorption spectra was 2 min initially. It was increased up to 10 min toward the end of photolysis. The final spectrum was taken after 30 min of irradiation. During the photolysis, the absorbance at 280 nm decreased; two new absorbance maxima at 248 and 305 nm are formed. 2. Incubate the reaction mixture for 10 min at 37°C in the dark. 3. Expose the sample to UV irradiation (see Notes 15 and 16). The irradiation times can be chosen in a range from 1 s to 60 min (see Note 17). 4. Keep the solutions in the dark before and after photolysis (see Note 6).

3.5. Analysis of DNA-Protein Adducts Analysis of DNA–protein adducts can be made, for example, by polyacrylamide gel electrophoresis of the irradiated samples followed by autoradio-

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graphy. SDS–polyacrylamide gel electrophoresis should be performed immediately after photocrosslinking according to Laemmli (24) with some variations. After the addition of 20 mg/mL of bromophenol blue, the samples are loaded onto a SDS–polyacrylamide gel of 5% polyacrylamide (separating gel) with an overlay of 3.5% polyacrylamide (stacking gel) containing 1% SDS. After the electrophoretic separation, the gels are silver-stained according to Adams and Sammons (25), dried, and exposed to X-ray film at –70°C. A quantitative determination of the DNA–protein adducts is possible by densitometric measurement of the autoradiogram (26). Figure 7 shows a typical result on photocrosslinking of EcoRI-digested plasmid pWH106 with a specific interacting protein (Tet repressor). Another possibility to detect the DNA–protein adducts is the application of the nitrocellulose filter binding assay according to Braun and Merrick (27), see also Chapter 1. 4. Notes 1. Experiments to synthesize [α-32P] or [U-14C]-labeled 8-N3dATP by starting the synthesis with [α-32P] or [U-14C]dATP, respectively, failed. This is most probably because of the formation of bromine radicals induced by radiation. These radicals could react unspecifically with the deoxyribonucleotide, suppressing the very specific electrophilic substitution of the hydrogen in position 8 of the adenine ring by the bromine ion. 2. Do not stop the reaction of dATP with bromine before 6 h even if the absorption maximum is near 262 nm after 1 or 2 h, otherwise a significant reduction of the yield of 8-BrdATP may occur. 3. 8-BrdATP obtained as triethylammonium salt is soluble in dimethylformamide in contrast to the alkali salts of this nucleotide. This is advantageous for the following substitution of bromine by the azido group yielding 8-N3dATP. 4. The exchange reaction of bromine by the azido group requires absolute dryness. However, the formation of 8-N3 dAMP and 8-N3dADP is usually observed, resulting from a limited hydrolytic cleavage of 8-N3dATP (see Fig. 5). 5. Besides the three azidoadenine deoxyribonucleotides, minor amounts of unreacted 8-bromoadenine deoxyribonucleotides are eluted as well (see Fig. 5). 6. Because of the photoreactivity of azido compounds, samples containing 8-N 3dATP should always be kept in the dark if possible. However, short exposure of azido compounds to normal daylight in our laboratory never falsified the results obtained. 7. 8-N3dATP can be stored frozen at –20°C in aqueous solution, pH 7.0, in the dark for at least 2 yr without significant loss of photoreactivity, as demonstrated by subsequent photocrosslinking experiments. 8. Exclude dithiothreitol from any buffers or other solutions that contain 8-N3dATP. It is well-known that dithiothreitol reduces azido groups to the corresponding amines (28). In addition, the UV absorbance of dithiothreitol resembles that


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Fig. 7. Photocrosslinking of proteins to DNA (pWH106). Autoradiogram of a denaturing 5% SDS-polyacrylamide gel electrophoresis showing photocrosslinking of Tet repressor to azido-activated 187-bp and 3848-bp fragments of pWH106 (radioactive labeled by 32P). Each 187-bp fragment contains two tet operator sequences, the 3848bp fragment none. UV irradiation of azidomodified 187-bp fragment in the presence of Tet repressor results in a reduced migration of the 187-bp fragment because of covalent crosslinking of the DNA to one or two Tet repressor dimers. In each of lanes 1–7, 0.06 pmol pWH106 (cleaved by EcoRI) and 20 pmol Tet repressor were applied. Lane 1: photoactive fragments of pWH 106 without protein (30' UV); lane 2: nonphotoactive fragments of pWH106 with Tet repressor (30' UV); lanes 3–7: photoactive fragments of pWH106 with Tet repressor (0', 1', 4', 10', 30' UV). Fractions: Origin of sample loading (a); traces of 3848-bp fragment covalently crosslinked (unspecifically) to Tet repressor (b); 3848-bp fragment (no Tet repressor bound) (c); 187-bp fragment covalently crosslinked to two Tet repressor dimers (d); 187-bp fragment covalently crosslinked to one Tet repressor dimer (e); 187-bp fragment (no Tet repressor bound) (f). of 8-N 3 dATP because of the formation of disulfide bonds by oxidation on storage in aqueous solution. This results in a reduced rate of photocrosslinking by the UV irradiation. 9. The UV absorption of 8-N3dATP shows a maximum at 280 nm. The absorbance at 280 nm increases with decreasing pH value (see step 2 of Subheading 3.2.). A second absorption maximum at 219 nm shifts to 204 nm in acidic solution. Both effects are a result of the protonation at N1 of the purine ring (29). The UV absorption spectrum of 8-N 3dATP resembles that of 8-N3ATP (23).

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Fig. 8. Conformation of adenine nucleotides. (From ref. 23.) 10. When testing its photoreactivity, take into account that the photolysis of 8-N3dATP is pH dependent. Exhaustive irradiation in neutral solution yields new absorption maxima at 248 and 305 nm, whereas in acidic or basic solution, the destruction of the purine ring is observed, as indicated by the disappearance of the absorbance between 240 and 310 nm (data not shown). 11. By analogy with 8-azidoadenine nucleotides, 8-N3dATP should prefer the syn conformation (Fig. 8) as a result of the bulky substituent in position 8 of the purine ring (30). This, however, seems to be contradicted by our results indicating that DNA polymerase I (E. coli) accepts 8-N3dATP in the nick translation reaction (it has been suggested that this enzyme only interacts with 2-deoxynucleoside triphosphates in the anti conformation [31]). The discrepancy may be explained in two ways: First, the steric requirements for the binding of 8-N3dATP by DNA polymerase I are less restrictive than assumed (32) or second, 8-N3dATP interacts in the anti conformation with the binding site of the enzyme. This could be demonstrated for the interaction of 8-N3ATP with the F1 ATPase from mitochondria (33). 12. The preparation of azido-modified and [32P]-labeled DNA by nick translation is critically dependent on the ratio of DNA, DNase I, and DNA polymerase I in the reaction medium. High concentrations of DNase I, on the one hand, result in a very efficient incorporation rate of the azido-modified and radioactive labeled deoxynucleotides, but on the other hand, the degradation of the DNA probes by DNase I has to be evaluated. Application of too small amounts of DNase I results in inefficient incorporation of the photoactivatable deoxynucleotides and in an insufficient photocrosslinking to the interacting proteins during subsequent irradiation of the azido-modified DNA. 13. Tris-HCl buffer, 50 mM, pH 7.2, can be used instead of double-distilled water without any significant effect on the photocrosslinking efficiency. 14. The amount of protein planned to be photocrosslinked to photoreactive DNA can be varied over a wide range. Too high an excess of proteins, however, should be


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avoided because the absorbance maximum of proteins at 280 nm will lead to inefficient crosslinking rates. 15. One way to expose the samples to UV light is to deposit the probes (typically 30– 50 µL) in plastic wells (normally used for radioimmunoassay or enzyme-linked immunosorbent assay tests). The UV lamp is positioned directly above the samples; thus more than one probe can be irradiated simultaneously. 16. The emitted light of the UV lamp used for photocrosslinking should not contain light of shorter wavelengths than 300 nm because of the possibility of photodamaging DNA or protein. For example, the Mineralight handlamp UVSL 25 (long wave) emits UV light of mainly 366 nm. The small portion of UV light of wavelengths between 300 and 320 nm emitted allows the photoactivation of the azido group without any significant photodamage of DNA or protein. 17. Ultraviolet irradiation times for photocrosslinking can be chosen over a wide range (see step 3 of Subheading 3.4.). Optimal UV irradiation rates (flux per unit area) must be tested. In our experiments (using the Mineralight handlamp UVSL 25 fixed in a position resulting in a fluence rate of 4 J/m2/s at the position of the sample) DNA–protein adducts are first detectable after irradiation times of 10–30 s; irradiation periods longer than 15–20 min do not improve the yield of photocrosslink products.

Acknowledgments The authors thank Dr. Marianne Schüz (Wiesbaden) for editing the manuscript. This work was supported by the Bundesministerium für Forschung und Technologie (07QV8942) and by the Deutsche Forschungsgemeinschaft (Scha 344/1-3). References 1. Bayley, H. and Knowles, J. R. (1977) Photoaffinity labeling. Methods Enzymol. 46, 69–114. 2. Bayley, H. (1983) Photogenerated reagents in biochemistry and molecular biology, in Laboratory Techniques in Biochemistry, vol. 12 (Work, T. S. and Burdon, R. H., eds.), Elsevier, Amsterdam. 3. Schäfer, H.-J. (1987) Photoaffinity labeling and photoaffinity crosslinking of enzymes, in Chemical Modifications of Enzymes, Active Site Studies (Eyzaguirre, J., ed.), Ellis Horwood, Chichester, pp. 45–62. 4. Smith, K. C. (1962) Dose dependent decrease in extractability of DNA from bacteria (by UV-light). Biochem. Biophys. Res. Commun. 8, 157–163. 5. Shetlar, M. D. (1980) Crosslinking of proteins to nucleic acids by UV-light. Photochem. Photobiol. Rev. 5, 105–197. 6. Welsh, J. and Cantor, C. R. (1984) Protein–DNA crosslinking. Trends Biochem. Sci. 9, 505–508. 7. Ekert, B., Giocanti, N., and Sabattier, R. (1986) Study of several factors in RNA– protein crosslink formation induced by ionizing radiations within 70S ribosomes of E. coli MRE 600. Int. J. Radiat. Biol. 50, 507–525.

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8. Lesko, S. A., Drocourt, J. L., and Yang, S. U. (1982) DNA–protein and DNA interstrand crosslinks induced in isolated chromatin by H2O2. and Fe-EDTA-chelates. Biochemistry 21, 5010–5015. 9. Wedrychowsky, A., Ward, W. S., Schmidt, W. N., and Hnilica, L. S. (1985) Chromium-induced crosslinking of nuclear proteins and DNA. J. Biol. Chem. 260, 7150–7155. 10. Summerfield, F. W. and Tappel, A. L. (1984) Crosslinking of DNA in liver and testis of rats fed 1,3-propanediol. Chem. Biol. Interact. 50, 87–96. 11. Dose, K., Bieger-Dose, A., Martens, K.-D., Meffert, R., Nawroth, T., Risi, S., et al. (1987) Survival under space vacuum—biochemical aspects. Proc. 3rd Eur. Symp. Life Sci. Res. in Space (ESA SP–271), pp. 193–195. 12. Lin, S. Y. and Riggs, A. D. (1974) Photochemical attachment of lac repressor to bromodeoxyuridine-substituted lac operator by UV radiation. Proc. Natl. Acad. Sci. USA 71, 947–951. 13. Evans, R. K., Johnson, J. D., and Haley, B. E. (1986) 5-Azido-2'-deoxyuridine-5'triphosphate: a photoaffinity labeling reagent and tool for the enzymatic synthesis of photoactive DNA. Proc. Natl. Acad. Sci. USA 83, 5382–5386. 14. Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent. EMBO J. 9, 2197–2205. 15. Lee, D. K., Evans, R. K., Blanco, J., Gottesfeld, J., and Johnson, J. D. (1991) Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'deoxyuridine-substituted DNA. J. Biol. Chem. 266, 16,478–16,484. 16. Blatter, E. E., Ebright, Y. W., and Ebright, R. H. (1992) Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking. Nature 83, 650–652. 17. Willis, M. C., Hicke, B. J., Uhlenbeck, O. C., Cech, T. R., and Koch, T. H. (1993) Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins. Science 262, 1255–1257. 18. Hicke, B. J., Willis, M. C., Koch, T. H., and Cech, T. R. (1994) Telomeric protein– DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine. Biochemistry 33, 3364–3373. 19. Bartholomew, B., Braun, B. R., Kassavetis, G. A., and Geiduschek, E. P. (1994) Probing close DNA contacts of RNA–polymerase III transcription complexes with the photoactive nucleoside 4-thiodeoxythymidine. J. Biol. Chem. 269, 18,090–18,095. 20. Meffert, R. and Dose, K. (1988) UV-induced crosslinking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides. FEBS Lett. 239, 190–194. 21. Meffert, R., Rathgeber, G., Schäfer, H.-J., and Dose, K. (1990) UV-induced crosslinking of Tet repressor to DNA containing tet operator sequences and 8-azidoadenines. Nucleic Acids Res. 18, 6633–6636. 22. Bartholomew, B., Tinker, R. L., Kassavetis, G. A., and Geiduschek, E. P. (1995) Photochemical crosslinking assay for DNA tracking by replication proteins. Methods Enzymol. 262, 476–494.


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23. Schäfer, H.-J., Scheurich, P., and Dose, K. (1978) Eine einfache darstellung von 8-N3ATP: ein Agens zur Photoaffinitätsmarkierung von ATP-bindenden Proteinen. Liebigs Ann. Chem. 1978, 1749–1753. 24. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. 25. Adams, L. D. and Sammons, D. W. (1981) A unique silver staining procedure for color characterization of polypeptides. Electrophoresis 2, 155–165. 26. Westermeier, R., Schickle, H., Thesseling, G., and Walter, W. W. (1988) Densitometrie von gelelektrophoresen. GIT Labor-Medizin 4/88, 194–202. 27. Braun, A. and Merrick, B. (1975) Properties of UV-light-mediated binding of BSA to DNA. Photochem. Photobiol. 21, 243–247. 28. Staros, J. V., Bayley, H., Standring, D. N., and Knowles, J. R. (1978) Reduction of aryl azides by thiols: implications for the use of photoaffinity reagents. Biochem. Biophys. Res. Commun. 80, 568–572. 29. Koberstein, R., Cobianchi, L., and Sund, H. (1976) Interaction of the photoaffinity label 8-azido-ADP with glutamate dehydrogenase. FEBS Lett. 64, 176–180. 30. Vignais, P. V. and Lunardi, J. (1985) Chemical probes of the mitochondrial ATP synthesis and translocation. Annu. Rev. Biochem. 54, 977–1014. 31. Czarnecki, J. J. (1978) Ph.D. Thesis, University of Wyoming, Laramie, WY. 32. Englund, P. T., Kelly, R. B., and Kornberg, A. (1969) Enzymatic synthesis of DNA: binding of DNA to DNA polymerase. J. Biol. Chem. 244, 3045–3052. 33. Garin, J., Vignais, P. V., Gronenborn, A. M., Clore, G. M., Gao, Z., and Bäuerlein, E. (1988) 1H-NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1-ATPase. FEBS Lett. 242, 178–182.

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24 Site-Specific Protein–DNA Photocrosslinking Analysis of Bacterial Transcription Initiation Complexes Nikolai Naryshkin, Younggyu Kim, Qianping Dong, and Richard H. Ebright 1. Introduction 1.1. Site-Specific Protein-DNA Photocrosslinking In work carried out in collaboration with the laboratory of D. Reinberg (University of Medicine and Dentistry of New Jersey), we have developed a site-specific protein–DNA photocrosslinking procedure to define positions of proteins relative to DNA in protein–DNA and multiprotein–DNA complexes (1–3). The procedure has four parts (Fig. 1): 1. Chemical (4–6) and enzymatic (7) reactions are used to prepare a DNA fragment containing a photoactivatible crosslinking agent and an adjacent radiolabel incorporated at a single, defined DNA phosphate (with a 9.7 Å linker between the photoreactive atom of the crosslinking agent and the phosphorus atom of the phosphate, and with an approximately 11 Å maximum “reach” between potential crosslinking targets and the phosphorus atom of the phosphate). 2. The multiprotein–DNA complex of interest is formed using the site-specifically derivatized DNA fragment, and the multiprotein–DNA complex is ultraviolet (UV)-irradiated, initiating covalent crosslinking with proteins in direct physical proximity to the photoactivatible crosslinking agent. 3. Extensive nuclease digestion is performed, eliminating uncrosslinked DNA and converting crosslinked DNA to a crosslinked, radiolabeled 3–5 nucleotide “tag.” 4. The “tagged” proteins are identified.

The procedure is performed in systematic fashion, with preparation and analysis of at least 10 derivatized DNA fragments, each having the photoactivatible crosslinking agent incorporated at a single, defined DNA phosphate From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Fig. 1. Site-specific protein–DNA photocrosslinking (1–3). (A,B) Chemical and enzymatic reactions are used to prepare a full-length-promoter DNA fragment with a phenyl-azide photoactivatible crosslinking agent (R) and an adjacent radioactive phosphorus (*) incorporated at a single, defined site. Based on the chemistry of incorporation, the maximum distance between the site of incorporation and the photoreactive atom is 9.7 Å; the maximum distance between the site of incorporation and a crosslinked atom is approx 11 Å. (C) UV irradiation of the derivatized protein–DNA complex initiates crosslinking. Nuclease digestion eliminates uncrosslinked DNA and converts crosslinked, radiolabeled DNA to a crosslinked, radiolabeled 3–5 nucleotide “tag.”

(typically each second DNA phosphate—each 12 Å—on each DNA strand spanning the region of interest (1–3,8). The results of the procedure define the translational positions of proteins relative to the DNA sequence. Plotted on a three-dimensional representation of a DNA helix, the results also define the rotational orientations of proteins relative to the DNA helix axis, and the groove orientations of proteins relative to the DNA major and minor grooves (1–3,8).


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The procedure has been validated in experiments with three multiprotein– DNA complexes for which crystallographic structures are available (i.e., the TBP–DNA complex, the TBP-TFIIA-DNA complex, and the TBP–TFIIB– DNA complex (1,9–13). In each case, there was a one-to-one correspondence between sites at which strong crosslinking was observed and sites that in the crystallographic structure were within 11 Å of crosslinked proteins (1,9–13). The procedure also has been applied to multiprotein-DNA complexes for which crystallographic structures are not available (1–3,8), including a eukaryotic transcription complex containing 16 distinct polypeptides and having a molecular mass in excess of 800 kDa (the RNAPII–TBP–TFIIB–TFIIF–DNA complex [2]) and a eukaryotic transcription complex containing 27 distinct polypeptides and having a molecular mass in excess of 1700 kDa (the RNAPII– TBP–TFIIB–TFIIE–TFIIF–TFIIH–DNA complex [2a]). The procedure is related to a procedure developed by Geiduschek and co-workers (14–17; see also refs. 18–23), but offers important advantages. First, because the photoactivatible crosslinking agent is incorporated into DNA chemically, it can be incorporated at a single, defined site. (In the procedure of Geiduschek and co-workers, this is true only at certain DNA sequences.) Second, because the photoactivatible crosslinking agent is incorporated on the DNA phosphate backbone, it can be incorporated at any nucleotide: A, T, G, or C. Third, since the photoactivatible crosslinking agent is incorporated on the DNA phosphate backbone, it probes interactions both in the DNA minor groove and in the DNA minor groove.

1.2. Bacterial Transcription Initiation Complexes Escherichia coli RNA polymerase holoenzyme (RNAP) consists of two copies of an α-subunit (36.5 kDa), one copy of a β-subunit (151 kDa), one copy of a β'-subunit (155 kDa), and one copy of a σ-subunit (70.3 kDa for the principle σ subunit species, σ70) (24). RNAP is a molecular machine that carries out a complex series of reactions in transcription initiation (24–26). Formation of a catalytically competent transcription initiation complex involves three steps (24–26): 1. RNAP binds to promoter DNA, interacting solely with DNA upstream of the transcription start, to yield an RNAP–promoter closed complex (RP c; also referred to as RPc1). 2. RNAP then wraps promoter DNA around its circumference, capturing and interacting with DNA downstream of the transcription start, and RNAP undergoes a protein conformational change, clamping tightly onto DNA, to yield an RNAP– promoter intermediate complex (RPi; also referred to as RPc2 and I2). 3. RNAP then melts approx 14 bp of promoter DNA surrounding the transcription start, rendering accessible the genetic information in the template strand of DNA, to yield a catalytically competent RNAP–promoter open complex (RPo).

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In the case of the E. coli lacPUV5 promoter, the RNAP–promoter intermediate complex (RPi) and the RNAP–promoter open complex (RPo) can be trapped by formation at 14–19°C in the absence of NTPs, and formation at 37°C in the absence of NTPs, respectively (27,28). Electrophoretic mobility shift, DNA footprinting, fluorescence anisotropy, and 2-aminopurine fluorescence experiments suggest that the trapped complexes are stable and homogeneous (28–31; A. Kapanidis, X. Shao, N.N., Y.K., and R.H.E., unpublished data). Kinetic experiments suggest that the trapped complexes correspond to bona fide on-pathway intermediates (27,28). In current work, we are using systematic site-specific protein–DNA photocrosslinking to define RNAP–promoter interactions in RNAP–promoter intermediate and open complexes. We are constructing a set of 110 derivatized DNA fragments, each containing a photoactivatible crosslinking agent incorporated at a single, defined position of the lacPUV5 promoter (positions –79 to +30). For each derivatized DNA fragment, we are forming RNAP–promoter intermediate and open complexes, isolating complexes using nondenaturing polyacrylamide gel electrophoresis, UV-irradiating complexes in situ—in the gel matrix—and identifying crosslinked polypeptides. We are performing experiments both with wild-type RNAP and with RNAP derivatives having discontinuous β and β' subunits (“split-β RNAP” and “split-β' RNAP;” reconstituted in vitro from recombinant α, recombinant σ70, and sets of recombinant fragments of β and β'; 32,33). Use of split-β and split-β' RNAP permits unambiguous assignment of crosslinks to β and β' (which are not well-resolved in SDS–polyacrylamide gel electrophoresis) and permits rapid, immediate mapping of crosslinks to segments of β and β' (e.g., N-terminal segment, central segment, or C-terminal segment) (Fig. 2). In this chapter, we present protocols for preparation of derivatized lacPUV5 promoter DNA fragments, formation of RNAP–promoter intermediate and open complexes, UV irradiation of complexes, and identification of crosslinks. In addition, we present support protocols for preparation of wild-type RNAP, split-β RNAP, and split-β' RNAP. 2. Materials 2.1. Preparation of Derivatized DNA Fragment, Chemical Reactions 1. 2. 3. 4. 5.

Azidophenacyl bromide (Sigma). Tetraethylthiuram disulfide/acetonitrile (PE Biosystems). dA-CPG, dC-CPG, dG-CPG, T-CPG (1 µmol, 500 Å) (PE Biosystems). dA, dC, dG, T β-cyanoethylphosphoramidites (PE Biosystems). Reagent kit for oligodeoxyribonucleotide synthesis (0.02 M iodine) (PE Biosystems).


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Fig. 2. Use of split-subunit RNAP derivatives (32,33) permits unambiguous assignment of crosslinks to RNAP subunits and permits rapid mapping of crosslinks to segments of RNAP subunits. (A) Subunit compositions of RNAP, two split-β RNAP derivatives, and two split-β' RNAP derivatives (idealized Coomassie-stained SDSPAGE gels). (B) Results of site-specific protein–DNA photocrosslinking experiments using the RNAP derivatives of panel A and a DNA fragment derivatized at a site close to or in contact with residues 1–235 of β, residues 821–1407 of β', and σ70 in the RNAP–promoter complex (idealized autoradiographs of SDS-PAGE gels).

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6. Denaturing loading buffer: 0.3% bromophenol blue, 0.3% xylene cyanol, and 12 mM EDTA, in formamide. 7. 0.5X TBE: 45 mM Tris-borate, pH 8.3, and 1 mM EDTA. 8. TE: 10 mM Tris-HCl, pH 7.6, 1 mM EDTA. 9. 50 mM triethylammonium acetate, pH 7.0 (Prime Synthesis). 10. 1 M potassium phosphate, pH 7.0. 11. 3 M sodium acetate, pH 5.2. 12. 100% ethanol (store at –20°C). 13. 70% ethanol (store at –20°C). 14. Dichloromethane (anhydrous) (PE Biosystems). 15. Acetonitrile (anhydrous) (PE Biosystems). 16. Acetonitrile (high-performance liquid chromatographic [HPLC] grade) (Fisher). 17. Formamide (Sigma). 18. 12% polyacrylamide (29:1 acrylamide:bis-acrylamide), 8 M urea, 0.5X TBE slab gel (10 × 7 × 0.075 cm). 19. Oligonucleotide purification cartridge (OPC) (PE Biosystems). 20. LiChrospher 100 RP–18 reversed-phase HPLC column (5 µm) (Merck). 21. Autoradiography intensifying screen (Sigma). 22. 254-nm germicidal lamp. 23. ABI392 DNA/RNA synthesizer (PE Biosystems). 24. Varian 5000 HPLC (Varian). 25. L-3000 diode-array HPLC UV detector (Hitachi). 26. Speedvac evaporator (Savant).

2.2. Preparation of Derivatized DNA Fragment, Enzymatic Reactions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Derivatized oligodeoxyribonucleotide (Subheading 3.1.). M13mp2(ICAP-UV5) or M13mp2(ICAP-UV5)-rev ssDNA (see Notes 1 and 2). T4 polynucleotide kinase (10 U/µL) (New England Biolabs, cat. no. M0201L). T4 DNA polymerase (3 U/µL) (New England Biolabs, cat. no. M0203L). T4 DNA ligase (5 U/µL) (Roche Molecular Biochemicals, cat. no. 799009). HaeIII (40 U/µL)(Roche Molecular Biochemicals, cat. no. 1336029). PvuII (40 U/µL)(Roche Molecular Biochemicals, cat. no. 899216). [γ32P]-ATP (10 mCi/mL, 6000 Ci/mmol) (NEN). 100 mM ATP (Amersham Pharmacia Biotech). 100 mM dNTPs (Amersham Pharmacia Biotech). Upstream primer (5'-CGGTGCGGGCCTCTTCGCTATTAC–3'). 10X phosphorylation buffer: 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, 15 mM β-mercaptoethanol. 13. 10X annealing buffer: 400 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 100 mM MgCl2. 14. 10X digestion buffer: 100 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 100 mM MgCl2, (see Note 3). 15. Elution buffer: 0.5 M ammonium acetate, 10 mM magnesium acetate pH 7.5, and 1 mM EDTA.


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16. Denaturing loading buffer: 0.3% bromophenol blue, 0.3% xylene cyanol, and 12 mM EDTA, in formamide. 17. Nondenaturing loading buffer: 0.3% bromophenol blue, 0.3% xylene cyanol, and 30% glycerol, in water. 18. 0.5X TBE: 45 mM Tris-borate pH 8.3, and 1 mM EDTA. 19. TE: 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. 20. Low-EDTA TE: 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. 21. 0.5 M EDTA, pH 8.0. 22. 10% sodium dodecyl sulfate (SDS). 23. 100% ethanol (store at –20°C). 24. 70% ethanol (store at –20°C). 25. 12% polyacrylamide (29:1 acrylamide:bis-acrylamide), 8 M urea, and 0.5X TBE slab gel (10 × 7 × 0.075 cm). 26. 7.5% polyacrylamide (29:1 acrylamide:bis-acrylamide), and 0.5X TBE slab gel (10 × 7 × 0.15 cm). 27. CHROMA SPIN+TE–10 spin column (Clontech). 28. CHROMA SPIN+TE–100 spin column (Clontech). 29. Spin-X centrifuge filter (0.22 µm, cellulose acetate) (Fisher). 30. PicoGreen dsDNA quantitation kit (Molecular Probes, cat. no. P-7589). 31. Disposable scalpels (Fisher). 32. Autoradiography markers (Stratagene). 33. Light box (VWR). 34. Speedvac evaporator (Savant).

2.3. Preparation of RNAP and RNAP Derivatives 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

E. coli strain XL1-blue (Stratagene, cat. no. 200249). E. coli strain BL21(DE3) pLysS (Novagen, cat. no. 69388-3). Plasmids encoding RNAP subunits (see Table 1). Plasmids encoding fragments of RNAP subunits (see Table 2). LB broth: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl; autoclave sterilized. TYE agar plates containing 200 µg/mL ampicillin and 35 µg/mL chloramphenicol: 10 g/L tryptone, 5 g/L yeast extract, 8 g/L NaCl, and 15 g/L agar; autoclave sterilized without antibiotics; supplemented with antibiotics after cooling to 55°C; poured into sterile 100 × 15 mm Petri plates at approx 25 mL/plate. TYE agar plates containing 200 µg/mL ampicillin and 20 µg/mL tetracycline. TYE agar plates containing 40 µg/mL kanamycin and 35 µg/mL chloramphenicol. TYE agar plates containing 40 µg/mL kanamycin and 20 µg/mL tetracycline. 100 mg/mL ampicillin (filter sterilized) (Sigma). 35 mg/mL chloramphenicol in ethanol (filter sterilized) (Sigma). 40 mg/mL kanamycin (filter sterilized) (Sigma). 20 mg/mL tetracycline in methanol (filter sterilized) (Sigma). 1 M IPTG (filter sterilized) (Roche Molecular Biochemicals). Buffer A: 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 5 mM imidazole. Buffer B: 20 mM Tris-HCl, pH 7.9, 6 M guanidine chloride, and 500 mM NaCl.

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Table 1 Plasmids Encoding RNAP Subunits Plasmid

Relevant Characteristics

pHTT7f1-NHα pMKSe2 pT7β' pHTT7f1-σ

ApR;

φ10P-rpoA(H6,Nter)a

ori-pBR322; ori-f1; ApR; ori-pBR322; lacP-rpoB ApR; ori-pBR322; φ10P-rpoC ApR; ori-pBR322; ori-f1; φ10P-rpoD

Ref. 34 35 36 34

arpoA(H6,Nter) is a derivative of rpoA having a nonnative hexahistidine coding sequence immediately after the rpoA start codon.

Table 2 Plasmids Encoding Fragments of RNAP Subunits Plasmid pβ1–235 pβ235–1342 pβ1–989 pβ951–1342 pβ'1–580 pβ'545–1407 pβ'1–878 pβ'821–1407

Relevant characteristics ApR Km R; ori-pBR322; lacP-rpoB(1–235) ApR; ori-pBR322; lacP-rpoB(235–1342) ApR; ori-pBR322; lacP-rpoB(1–989) ApR; ori-pBR322; φ10P-rpoB(950–1342) ApR; ori-pBR322; ori-f1; lacP-φ10P-rpoC(1–580) ApR; ori-pBR322; ori-f1; lacP-φ10P-rpoC(545–1407) ApR; ori-pBR322; ori-f1; lacP-φ10P-rpoC(1–878) KmR; ori-pBR322; ori-f1; φ10P-rpoC(821–1407)

Ref. 32 32 32 32 33 33 33 33

17. Buffer C: 40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT). 18. Buffer D: 50 mM Tris-HCl, pH 7.9, 6 M guanidine chloride, 10 mM MgCl2 , 0.01 mM ZnCl2, 1 mM EDTA, 10 mM DTT, and 10% glycerol. 19. Buffer E: 50 mM Tris-HCl, pH 7.9, 200 mM KCl, 10 mM MgCl2, 0.01 mM ZnCl2, 1 mM EDTA, 5 mM β-mercaptoethanol, and 20% glycerol. 20. Buffer F: 50 mM Tris-HCl, pH 7.9, and 5% glycerol. 21. α Storage buffer: 50 mM Tris-HCl, pH 7.9, 200 mM KCl, 10 mM MgCl2, 1 mM EDTA, 5 mM β-mercaptoethanol, and 20% glycerol. 22. 2X SDS loading buffer: 63 mM Tris-HCl, pH 8.3, 2% SDS, 5% β-mercaptoethanol, 25% glycerol, and 0.3% bromophenol blue. 23. SDS running buffer: 25 mM Tris, 250 mM glycine pH 8.3, and 0.1% SDS. 24. Destaining solution: 10% acetic acid, 50% methanol, and 40% water. 25. 100 mM PMSF in ethanol (Sigma). 26. 2% lysozyme (Sigma, cat. no. L-6876) (approx 50,000 U/mg). 27. 10% sodium deoxycholate (Sigma). 28. 10% n-octyl-β-D-glucopyranoside (Sigma). 29. Triton X-100 (Sigma).

Protein–DNA Photocrosslinking 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

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2 M imidazole (pH adjusted to 8.0 with 10 M HCl) (Sigma). Glycerol (Fisher). Trichloroacetic acid (Aldrich). Coomassie Brilliant Blue G-250 (Bio-Rad). Acetone (Aldrich). 10% polyacrylamide (37.5:1 acrylamide:bis-acrylamide), and 0.1% SDS, slab gel (10 × 7 × 0.075 cm). Prestained protein molecular-weight markers (7-210 kDa) (Bio-Rad). Bio-Rad Protein Assay Kit (cat. no. 500-0002). Ni:NTA-agarose (Qiagen). Dialysis membranes (10-kDa molecular-weight cutoff) (VWR, cat. no. 25223-821). Dialysis-membrane closures (VWR). Collodion dialysis bags (10-kDa molecular-weight cutoff) (Schleicher & Schuell). Nanosep-30K centrifugal concentrators (VWR). Econo-Pac 20-mL chromatography columns (Bio-Rad). Chromatography column frits (1.5 × 0.3 cm) (Bio-Rad). 15 mL culture tubes (autoclave-sterilized) (VWR). Culture-tube stainless-steel closures (autoclave sterilized) (VWR). 2.8-L triple-baffled Fernbach flask (autoclave sterilized) (Bellco Glass, cat. no. 2551-02800). 30-mL polypropylene copolymer centrifuge tube with cap (VWR, cat. no. 21010-567). 250-mL polypropylene copolymer centrifuge bottle with cap (VWR, cat. no. 21020-028). 1-L polypropylene copolymer centrifuge bottle with cap (VWR, cat. no. 21020-061). 200-mL steel beaker (VWR). Branson 450 sonicator (VWR). Sorvall RC-3B centrifuge (DuPont). Sorvall RC-5B centrifuge (DuPont).

2.4. In-Gel Photocrosslinking 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Cystamine dihydrocloride (Sigma). Acryloyl chloride (Aldrich). Acrylamide (Bio-Rad). TEMED (Bio-Rad). 10% ammonium persulfate (freshly made). SurfaSil siliconizing agent (Pierce). Derivatized promoter DNA fragment (Subheading 3.2.). RNAP or RNAP derivative (Subheading 3.3.). DNase I (126 units/µL) (Sigma, cat. no. D7291). Micrococcal nuclease in nuclease dilution solution (50 U/µL) (Pharmacia, cat no. 27-0584). 11. Nuclease dilution solution: 5 mM CaCl2, 0.1 mM PMSF, and 50% glycerol.

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12. 2X DTT-free transcription buffer: 50 mM HEPES–HCl, pH 8.0, 200 mM KCl, 20 mM MgCl2, and 10% glycerol. 13. Nondenaturing loading buffer: 0.3% bromophenol blue, 0.3% xylene cyanol, and 30% glycerol. 14. 5X SDS loading buffer: 300 mM Tris-HCl, pH 8.3, 10% SDS, 20 mM EDTA, 25% β-mercaptoethanol, 0.1% bromophenol blue, 50% glycerol. 15. 0.5X TBE: 45 mM Tris-borate, pH 8.0, and 1 mM EDTA. 16. SDS running buffer: 25 mM Tris, 250 mM glycine, pH 8.3, and 0.1% SDS. 17. 10% SDS. 18. 1 M DTT (freshly made). 19. 0.2 mM PMSF (Sigma). 20. 0.22 mg/mL heparin (Sigma, cat. no. H-3393) (grade I-A, from porcine intestinal mucosa, approx 170 USP units/mg). 21. 4–15% gradient polyacrylamide (37.5:1 acrylamide:bis-acrylamide) Tris–HCl slab gel (Bio-Rad, cat. no. 161-1176). 22. Prestained protein molecular-weight markers (7–210 kDa) (Bio-Rad). 23. Silicone rubber heating mat (200 W, 120 V AC; 25 × 10 cm) (Cole-Parmer, cat. no. P-03125-40). 24. Variable voltage controller (Cole-Parmer, cat. no. P-01575-10). 25. Digital thermometer (Cole-Parmer, cat. no. P-91000-00). 26. Thermocouple probe (needle, 0.7 mm in diameter) (Cole-Parmer, cat. no. P-91000-00). 27. Large binder clips (5-cm width) (Staples). 28. Filter unit (22-µm pore size, 250 mL) (Millipore). 29. 50-mL Büchner funnel with glass frit (10 µm pore size) (Fisher). 30. 500-mL separating funnel (Fisher). 31. Disposable scalpels (VWR). 32. X-ray exposure holder with intensifying screen (Kodak). 33. Light box (VWR). 34. Rayonet RPR-100 photochemical reactor equipped with 16 RPR-3500 Å tubes (Southern New England Ultraviolet). 35. Speedvac evaporator (Savant).

3. Methods 3.1. Preparation of Derivatized DNA Fragment, Chemical Reactions

3.1.1. Preparation of Phosphorothioate Oligodeoxyribonucleotide 1. Perform 24 standard cycles of solid-phase β-cyanoethylphosphoramidite oligodeoxyribonucleotide synthesis to prepare CPG-linked precursor containing residues 3–26 of desired oligodeoxyribonucleotide. Use the following settings: cycle, 1.0 µM CE; DMT, on; end procedure, manual. 2. Replace iodine/water/pyridine/tetrahydrofuran solution (bottle 15) by tetraethylthiuram disulfide/acetonitrile solution. Perform one modified cycle of solid-


3.

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phase β-cyanoethylphosphoramidite oligodeoxyribonucleotide synthesis to add residue 2 and phosphorothioate linkage. Use the following settings: cycle, 1.0 mM sulfur; DMT, on; end procedure, manual. Replace tetraethylthiuram disulfide/acetonitrile solution (bottle 15) by iodine/ water/pyridine/tetrahydrofuran solution. Place collecting vial on the DNA synthesizer. Perform one standard cycle of solid-phase β-cyanoethylphosphoramidite oligodeoxyribonucleotide synthesis to add residue 1. Use the following settings: cycle, 1.0 µM CE; DMT, on; end procedure, CE. Remove collecting vial, screw cap tightly, and deblock by incubating 8 h at 55°C. Transfer sample to 6-mL polypropylene round-bottomed tube, place tube in Speedvac, and spin 20 min with Speedvac lid ajar and with no vacuum (allowing evaporation of ammonia). Close Speedvac lid, apply vacuum, and dry. Detritylate and purify approx 0.075 µmol on OPC according to supplier’s protocol. Dry in Speedvac. Resuspend in 100 µL TE. Remove 2-µL aliquot, dilute with 748 µL TE, and determine concentration from UV absorbance at 260 nm (molar extinction coefficient = 240,000 AU/M/cm). To confirm purity of oligodeoxyribonucleotide, mix aliquot containing 1 nmol oligodeoxyribonucleotide with equal volume of formamide. Apply to 12% polyacrylamide (29:1 acrylamide:bis-acrylamide), 8 M urea, 0.5X TBE slab gel (10 × 7 × 0.075 cm). As marker, load in adjacent lane 5 mL denaturing loading buffer. Electrophorese 30 min at 25 V/cm. Disassemble gel, place on intensifying screen, and view in dark using 254 nm germicidal lamp. Oligodeoxyribonucleotide should appear as dark shadow against green background and should migrate more slowly than bromophenol blue. If purity is ≥95%, proceed to next step. Divide remainder of sample into 50-nmol aliquots, transfer to 1.5-mL siliconized polypropylene microcentrifuge tubes, dry in Speedvac, and store at –20°C (stable for at least 2 yr).

3.1.2. Derivatization of Oligodeoxyribonucleotide (All Steps Carried Out Under Subdued Lighting [see Note 4]) 1. Dissolve 10 mg (42 µmol) azidophenacyl bromide in 1 mL chloroform. Transfer 100-µL aliquots (4.2 µmol) to 1.5-mL siliconized polypropylene microcentrifuge tubes, and dry in Speedvac. Wrap tubes with aluminum foil, and store desiccated at 4°C (stable indefinitely). 2. Resuspend 50-nmol aliquot of phosphorothioate oligodeoxyribonucleotide (Subheading 3.1.1.) in 50 µL water, and resuspend 4.2-µmol aliquot of azidophenacyl bromide in 220 µL methanol. 3. Mix 50 µL (50 nmol) phosphorothioate oligodeoxyribonucleotide solution, 5 µL 1 M potassium phosphate (pH 7.0), and 55 µL (1 µmol) azidophenacyl bromide solution in a 1.5-mL siliconized polypropylene microcentrifuge tube. Incubate 3 h at 37°C in the dark. 4. Precipitate derivatized oligodeoxyribonucleotide by adding 11 µL of 3 M sodium acetate (pH 5.2) and 275 µL ice-cold 100% ethanol. Invert tube several times,

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3.1.3. Purification of Derivatized Oligodeoxyribonucleotide (All Steps Carried Out Under Subdued Lighting [see Note 4]) 1. Resuspend derivatized oligodeoxyribonucleotide in 100 µL of 50 mM triethylammonium acetate (pH 7.0). 2. Analyze 5-µL aliquot by C18 reversed-phase HPLC to confirm efficiency of derivatization reaction. Use LiChrospher 100 RP-18 C18 reversed-phase HPLC column (5 µm), with solvent A = 50 mM triethylammonium acetate (pH 7.0) and 5% acetonitrile; solvent B = 100% acetonitrile; and flow rate = 1 mL/min. Equilibrate column with 10 column volumes of solvent A before loading sample. After loading sample, wash column with 6 column volumes of solvent A and elute with a 50 min gradient of 0–70% solvent B in solvent A. Derivatized and underivatized oligodeoxyribonucleotides elute at approx 25% solvent B and approx 16% solvent B, respectively (see Notes 5 and 6). 3. If derivatization efficiency is ≥80%, purify remainder of sample using procedure in step 2, collecting peak fractions (see Notes 5 and 6). 4. Pool peak fractions, divide into 1-mL aliqouts, and dry in Speedvac. Store desiccated at –20°C in the dark (stable for at least 1 yr). 5. Resuspend one aliquot in 100 µL TE. Remove 5 µL, dilute with 495 µL water, and determine concentration from UV absorbance at 260 nm (molar extinction coefficient = 242,000 AU/M/cm). 6. Divide remainder of derivatized-oligodeoxyribonucleotide/TE solution from step 5 into 20, 5-pmol aliquots and one larger aliquot, dry in Speedvac, and store desiccated at –20°C in the dark (stable for at least 1 yr).

3.2. Preparation of Derivatized DNA Fragment, Enzymatic Reactions 3.2.1. Radiophosphorylation of Derivatized Oligodeoxyribonucleotide (All Steps Carried Out Under Subdued Lighting [see Note 4]) 1. Resuspend 5 pmol derivatized oligodeoxyribonucleotide in 12 µL water. Add 2 µL of 10X phosphorylation buffer, 5 µL of [γ-32P]ATP (50 µCi) and 1 µL (10 U) T4 polynucleotide kinase. Incubate 15 min at 37°C. Terminate reaction by heating 5 min at 65°C (see Note 7). 2. Add 15 µL water. 3. Desalt radiophosphorylated derivatized oligodeoxyribonucleotide into TE using CHROMA SPIN+TE–10 spin column according to supplier’s protocol. 4. Immediately proceed to next step, or, if necessary, store radiophosphorylated derivatized oligodeoxyribonucleotide solution at –20°C in the dark (stable for up to 24 h).

3.2.2. Annealing, Extension, and Ligation of Radiophosphorylated Derivatized Oligodeoxyribonucleotide (All Steps Carried Out Under Subdued Lighting [see Note 4]) 1. In 1.5-mL siliconized polypropylene microcentrifuge tube, mix 34 µL radiophosphorylated derivatized oligodeoxyribonucleotide, 1 µL of 10 µM upstream


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primer, 1 µL of 1 µM M13mp2(ICAP-UV5) ssDNA (for analysis of crosslinks to template DNA strand) or M13mp2(ICAP-UV5)-rev ssDNA (for analysis of crosslinks to nontemplate DNA strand), and 4 µL of 10X annealing buffer. Heat 5 min at 65°C (see Note 7). Transfer to 500-mL beaker containing 200 mL water at 65°C, and place beaker at room temperature to permit slow cooling (65°C to 25°C in approx 60 min). Add 2 µL of 25 mM dNTPs, 1 µL of 100 mM ATP, 3 µL (9 units) T4 DNA polymerase, and 1 µL (5 units) T4 DNA ligase. Perform parallel reaction without ligase as “no-ligase” control. Incubate 15 min at room temperature, followed by 3 h at 37°C. Terminate reaction by adding 1 µL of 10% SDS. Desalt into TE using CHROMA SPIN+TE-100 spin column according to supplier’s protocol. Immediately proceed to next step.

3.2.3. Digestion and Purification of Derivatized DNA Fragment (All Steps Carried Out Under Subdued Lighting [see Note 4]) 1. In 1.5-mL siliconized polypropylene microcentrifuge tube, mix 40 µL product from Subheading 3.2.2., 4.5 µL of 10X digestion buffer, 0.25 µL (10 units) HaeIII or 0.25 µL (10 units) PvuII (see Note 8). Incubate 1 h at 37°C. 2. Perform parallel reaction using 40 µL “no-ligase” control from step 3 of Subheading 3.2.2. 3. Mix 3 µL aliquots of reaction of step 1 and of “no-ligase” control reaction of step 2, each with 7 µL denaturing loading buffer. Heat 5 min at 65°C, and then apply to 12% polyacrylamide (29:1 acrylamide:bis-acrylamide), 8 M urea, 0.5X TBE slab gel (10 × 7 × 0.075 cm). As a marker, load 5 µL denaturing loading buffer in the adjacent lane. Electrophorese 30 min at 25 V/cm. Dry gel, expose to X-ray film 1 h at room temperature, and process film. Estimate ligation efficiency by comparing reaction and “no-ligase” control lanes. If the ligation efficiency is ≥80%, proceed to the next step. If not, repeat the steps of Subheadings 3.2.1. and 3.2.2. 4. Mix remainder of reaction of step 1 (42 µL) with 10 µL 50% glycerol. Apply to nondenaturing 7.5% polyacrylamide (29:1 acrylamide:bis-acrylamide), 0.5X TBE slab gel (10 × 7 × 0.15 cm). As a marker, load 5 µL nondenaturing loading buffer in the adjacent lane. Electrophorese at 25 V/cm until the bromophenol blue reaches the bottom of the gel. 5. Remove one glass plate, and cover the gel with plastic wrap. Attach two autoradiography markers to the gel. Expose to X-ray film for 60 s at room temperature and process the film. Cut out the portion of the film corresponding to the derivatized DNA fragment. Using a light box, superimpose the cut-out film on the gel, using autorad markers as the alignment reference points. Using disposable scalpel, excise portion of gel corresponding to derivatized DNA fragment. 6. Place the excised gel slice in a 1.5-mL siliconized polypropylene microcentrifuge tube, and crush with a 1-mL pipet tip. Add 300 µL elution buffer, centrifuge 5 s at 5000g, and incubate 12 h at 37°C. 7. Transfer supernatant to Spin-X centrifuge filter and centrifuge 1 min at 13,000g at room temperature in fixed-angle microcentrifuge.

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8. Transfer filtrate to a 1.5-mL siliconized polypropylene microcentrifuge tube. Precipitate the derivatized DNA fragment by the addition of 1 mL ice-cold 100% ethanol. Invert the tube several times and place at –20°C for 30 min. Centrifuge 5 min at 13,000g at 4°C in a fixed-angle microcentrifuge. Remove and dispose of supernatant, wash pellet with 500 µL ice cold 70% ethanol, and air-dry for 15 min at room temperature. 9. Resuspend in 30 µL Low-EDTA TE. Determine radioactivity by Cerenkov counting. Remove 1 µL aliquot, and determine DNA concentration using PicoGreen dsDNA quantitation kit according to supplier’s protocol. Calculate specific activity (expected specific activity ≈5,000 Ci/mmol). 10. Store derivatized DNA fragment at 4°C in the dark (stable for ≈1 wk).

3.3. Preparation of RNAP and RNAP Derivatives 3.3.1. Preparation of Hexahistidine-Tagged Recombinant α-Subunit 1. Transform E. coli strain BL21(DE3) pLysS with plasmid pHTT7f1-NHα. Plate to TYE agar containing 200 µg/mL ampicillin and 35 µg/mL chloramphenicol, and incubate 12 h at 37°C. 2. Inoculate single colony into 5 mL LB containing 200 µg/mL ampicillin and 35 µg/mL chloramphenicol in a 15-mL culture tube with a culture tube stainlesssteel closure, and shake vigorously for 12 h at 37°C. Transfer to a 15 mL polypropylene centrifuge tube, and centrifuge 5 min at 3000g at room temperature. Discard supernatant, wash cell pellet twice with 5 mL LB, and resuspend cell pellet in 5 mL LB in a new 15-mL polypropylene centrifuge tube. 3. Inoculate into 1 L LB containing 200 µg/mL ampicillin and 35 µg/mL chloramphenicol in a 2.8-L Fernbach flask, and shake vigorously at 37°C until OD600 = 0.6. Add 1 mL of 1 M IPTG, and shake vigorously for an additional 3 h at 37°C. 4. Transfer culture to a 1-L polypropylene copolymer centrifuge bottle. Harvest cells by centrifugation 20 min at 5000g at 4°C. 5. Resuspend cell pellet in 100 mL buffer A at 4°C. Transfer into a 200-mL steel beaker and place beaker on ice. Lyse cells with four 40-s sonication pulses at 25% maximum sonicator output (2-min pause between each pulse). 6. Transfer lysate to a 250-mL polypropylene copolymer centrifuge bottle. Centrifuge for 15 min at 15,000g at 4°C. Collect supernatant. 7. Transfer supernatant to a 250-mL glass beaker. Add 35 g ammonium sulfate and stir for 20 min on ice. 8. Transfer suspension to a 250-mL polypropylene copolymer centrifuge bottle. Centrifuge for 20 min at 15,000g at 4°C. Discard the supernatant. 9. Resuspend the pellet in 28 mL buffer B containing 5 mM imidazole. Transfer to a 30-mL polypropylene copolymer centrifuge tube and rock gently for 30 min at 4°C. Centrifuge for 15 min at 15,000g at 4°C. 10. Load the supernatant onto a 5-mL Ni:NTA–agarose column pre-equilibrated with 25 mL buffer B containing 5 mM imidazole (see Note 9). Collect flowthrough and reload onto column. Wash column with 50 mL buffer B containing 5 mM imidazole, and 25 mL buffer B containing 10 mM imidazole. Elute column with


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12.

13. 14.

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15 mL buffer B containing 20 mM imidazole, 15 mL buffer B containing 30 mM imidazole, 15 mL buffer B containing 40 mM imidazole, and 15 mL buffer B containing 150 mM imidazole. Collect 5-mL fractions. Transfer a 10-µL aliquot of each fraction to a 1.5-mL siliconized polypropylene microcentrifuge tube, add 90 µL water and 100 µL of 10% trichloroacetic acid. Place on ice for 20 min. Centrifuge for 5 min at 13,000g at room temperature. Discard the supernatant. Wash the pellet with 500 µL acetone and air-dry for 15 min. Dissolve the pellet in 5 µL water, add 5 µL of 2X SDS loading buffer, heat for 3 min at 100°C, and apply to 10% polyacrylamide (37.5:1 acrylamide:bis-acrylamide) and 0.1% SDS slab gel (10 × 7 × 0.075 cm). As a marker, load 5 µL prestained protein molecular weight markers into the adjacent lane. Electrophorese in SDS running buffer at 25 V/cm until the bromophenol blue reaches the bottom of gel. Stain the gel by gently shaking for 5 min in 50 mL of 0.2% Coomassie Brilliant Blue G–250 in the destaining solution. Destain by gently shaking for 10 h in 100 mL destaining solution. Pool fractions containing homogenous α (typically fractions with buffer B containing 40–150 mM imidazole). Dialyze using a 10-kDa molecular-weight-cutoff dialysis membrane against two 1-L changes of α storage buffer for 16 h at 4°C. Determine protein concentration and total protein amount using Bio-Rad Protein Assay according to the supplier’s protocol. After dialysis, measure the volume and transfer to a 30-mL polypropylene copolymer centrifuge tube. Add 3 g ammonium sulfate per 10 mL and rock gently for 20 min at 4°C. Centrifuge for 20 min at 15,000g at 4°C. Remove and discard 10 mL of supernatant. Resuspend pellet in the remaining supernatant. Divide into 50-µL aliquots and transfer to 1.5-mL siliconized polypropylene microcentrifuge tubes. Centrifuge aliquots for 5 min at 13,000g at 4°C. Store at –80°C (stable for at least 1 yr). Expected yield: 20–30 mg (250–500 µg/aliquot). Expected purity: >99%.

3.3.2. Preparation of Crude Recombinant RNAP Subunits and Subunit Fragments 1. Transform plasmid encoding RNAP subunit or subunit fragment into E. coli strain BL21(DE3) pLysS (for plasmids with φ10P- or lacP-φ10P-based expression; Tables 1 and 2) or E. coli strain XL1-blue (for plasmids with lacP-based expression; Tables 1 and 2). Plate transformants of BL21(DE3) pLysS to TYE agar containing 200 µg/mL ampicillin (40 µg/mL kanamycin for plasmid pβ'821–1407) and 35 µg/mL chloramphenicol, and incubate for 12 h at 37°C. Plate transformants of XL1-blue to TYE agar containing 200 µg/mL ampicillin (40 µg/mL kanamycin for plasmid pβ1–235) and 20 µg/mL tetracycline, and incubate for 16 h at 37°C. 2. Inoculate a single colony into 5 mL LB containing antibiotics at concentrations specified in step 1 in a 15-mL culture tube with stainless-steel closure, and shake vigorously for 12 h at 37°C. Transfer to a 15-mL polypropylene centrifuge tube and centrifuge for 5 min at 3000g at room temperature. Discard the supernatant,

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wash the cell pellet twice with 5 mL LB, and resuspend the cell pellet in 5 mL LB. 3. Inoculate into 1 L LB containing antibiotics at concentrations specified in step 1 in a 2.8-L Fernbach flask, and shake vigorously at 37°C until OD600 = 0.6. Add 1 mL of 1 M IPTG and shake vigorously for an additional 3 h [transformants of BL21(DE3) pLysS] or 5 h (transformants of XL1-blue) at 37°C. 4. Transfer culture to a 1 L polypropylene copolymer centrifuge bottle. Harvest the cells by centrifugation for 20 min at 5000g at 4°C. 5. Resuspend the cell pellet in 10 mL buffer C containing 0.2% sodium deoxycholate and 0.02% lysozyme in a 30-mL polypropylene copolymer centrifuge tube at 4°C. Place the tube on ice. Lyse cells with five 30-s sonication pulses at 25% maximum sonicator output (2-min pause between each pulse). 6. Centrifuge 20 min at 15,000g at 4°C. Discard the supernatant. 7. Resuspend the pellet in 10 mL buffer C containing 0.2% n-octyl-β-D-glucopyranoside (0.5% Triton X–100 for preparation of σ70) and 0.02% lysozyme at 4°C. Sonicate as in step 5. Centrifuge for 20 min at 15,000g at 4°C. Discard the supernatant. 8. Resuspend pellet in 10 mL buffer C containing 0.2% n-octyl-β-D-glucopyranoside (0.5% Triton X–100 for preparation of σ70) at 4°C. Sonicate as in step 5. Centrifuge for 20 min at 15,000g at 4°C. Discard the supernatant. 9. Resuspend pellet in 10 mL buffer C at 4°C. Place tube on ice and sonicate for 10 s at 25% maximum sonicator output. Divide into 500-µL aliquots and transfer to 1.5-mL siliconized polypropylene microcentrifuge tubes. Centrifuge for 5 min at 13,000g at 4°C. Discard the supernatant. 10. Add 100 µL ice-cold buffer C containing 10% glycerol to each aliquot. Store at –80°C (stable for at least 2 yr). Expected yield: 50–100 mg (1.5–3 mg/aliquot). Expected purity: 50–90%.

3.3.3. Reconstitution of RNAP and RNAP Derivatives 1. Thaw aliquots containing purified subunit (from Subheading 3.3.1., step 15) and crude recombinant RNAP subunits and subunit fragments (from Subheading 3.3.2., step 10) by placing on ice for 10 min. Centrifuge for 30 s at 13,000g at 4°C. Discard supernatants. 2. Resuspend each pellet in 500 µL buffer D. Incubate for 30 min at 4°C, rocking gently. Centrifuge for 5 min at 13,000g at 4°C. 3. Transfer supernatants to new 1.5-mL siliconized polypropylene microcentrifuge tubes at 4°C. Determine the protein concentrations using Bio-Rad Protein Assay according to the supplier’s protocol (expected concentrations: 3–6 mg/mL). 4. Prepare core reconstitution mixture by combining in a 1.5-mL siliconized polypropylene microcentrifuge tube the following: 30 µg N-terminally hexahistidine-tagged α, 300 µg β (or 170 µg β1–235 and 800 µg β235–1342; or 700 µg β1–989 and 300 µg β951–1342) and 500 µg β' (or 400 µg β'1–581 and 500 µg β'545–1407; or 700 µg β'1–877 and 330 µg β'821–1407), and diluting with buffer D to a total protein concentration of 450 µg/mL.


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5. Prepare σ70 reconstitution mixture by adding 250 µg σ70 to a 1.5-mL siliconized polypropylene microcentrifuge tube and diluting with buffer D to a total protein concentration of 1500 µg/mL. 6. Dialyze core and σ70 reconstitution mixtures separately in collodion dialysis bags against two 1-L changes of buffer E for 16 h at 4°C. 7. Transfer core and σ70 reconstitution mixtures to separate 2.0-mL siliconized polypropylene microcentrifuge tubes. Centrifuge for 5 min at 13,000g at 4°C. Combine supernatants in a single, new 2.0-mL siliconized polypropylene microcentrifuge tube. 8. Incubate 45 min at 30°C. Centrifuge for 10 min at 13,000g at 4°C.

3.3.4. Purification of RNAP and RNAP Derivatives 1. During incubation of step 8 of Subheading 3.3.3., place 200 µL Ni:NTA–agarose in a 2.0-mL siliconized polypropylene microcentrifuge tube and centrifuge for 2 min at 13,000g at 4°C. Remove supernatant. 2. Resuspend Ni:NTA–agarose in 1 mL buffer F containing 5 mM imidazole at 4°C. Centrifuge 2 min at 13,000 × g at 4°C. Remove supernatant. Repeat two times. 3. Add supernatant of step 8 of Subheading 3.3.3. to Ni:NTA–agarose from step 2. Incubate 45 min at 4°C, rocking gently. Centrifuge for 2 min at 13,000g at 4°C. Discard the supernatant. 4. Resuspend in 1.5 mL buffer F containing 5 mM imidazole at 4°C. Rock gently for 15 s at 4°C. Centrifuge 2 min at 13,000g at 4°C. Discard the supernatant. Repeat two times. 5. Resuspend in 250 µL buffer F containing 150 mM imidazole. Rock gently for 2 min at 4°C. Centrifuge for 2 min at 13,000g at 4°C. 6. Transfer supernatant to Nanosep-30K centrifugal concentrator. Centrifuge at 13,000g at 4°C until sample volume is reduced to approx 50 mL (approx 15 min). 7. Transfer the sample to a 1.5-mL siliconized polypropylene microcentrifuge tube. Add, in order, 1 µL of 0.1 M β-mercaptoethanol and 50 µL glycerol, mix well, and store at –20°C (stable for at least 1 mo). 8. Determine the protein concentration using the Bio-Rad Protein Assay according to the supplier’s protocol. Expected yield: 100 µg. Expected purity: >90%.

3.4. In-Gel Photocrosslinking 3.4.1. Synthesis of N,N'-Bisacryloylcystamine (BAC) (see Note 10) 1. Acryloyl chloride is highly toxic. Therefore, all manipulations in this section must be performed in a fume hood. 2. Dissolve 4.0 g (18 mmol) cystamine dihydrochloride in 40 mL of 3 M NaOH (120 mmol). Dissolve 4.3 mL (54 mmol) acryloyl chloride in 40 mL chloroform. Mix solutions in 500 mL flask (see Note 11). (Two phases will form: an upper, aqueous phase; and a lower, organic phase.) Place flask on a plate stirrer and stir 3 min at room temperature, followed by 15 min at 50°C. 3. Discontinue stirring. Immediately transfer reaction mixture to a 500-mL separating funnel, allow phases to separate (approx 2 min), and transfer lower, organic phase to a 250-mL beaker.

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4. Place on ice for 10 min. Collect precipitate by filtration in Büchner funnel. 5. Transfer precipitate to a 250-mL beaker with 30 mL chloroform at room temperature. Place beaker on plate stirrer and stir 1 min at room temperature, followed by 5 min at 50°C. Place on ice for 10 min and collect precipitate (BAC) by filtration in Büchner funnel. 6. Transfer precipitate to a 50-mL polypropylene centrifuge tube. Seal tube with Parafilm, pierce seal several times with a syringe needle, place tube in vacuum desiccator, and dry under a vacuum for 16 h at room temperature. Expected yield: 1.5–1.9 g.

3.4.2. Preparation of Polyacrylamide:BAC Gel 1. Prepare 20% acrylamide:BAC (19:1) stock solution by dissolving, in order, 19 g acrylamide and 1 g BAC in 80 mL water in a 200-mL beaker at room temperature. Place on a plate stirrer and stir for 10 min at 60°C (see Note 12). Adjust volume to 100 mL with water. Allow solution to cool to room temperature. Filter stock solution using 0.22-µm filter unit and store at room temperature in the dark (stable for at least 2 mo). 2. Mix 9 mL of 20% acrylamide:BAC (19:1) stock solution, 1.8 mL of 10X TBE, and 25.2 mL water. Add 180 µL TEMED and 90 µL freshly prepared 10% ammonium persulfate (see Note 13). Immediately pour into slab gel assembly with siliconized notched glass plate (27 × 16 × 0.1 cm) (see Note 14). Insert comb and heat slab gel assembly to approx 60°C by positioning a bench lamp with a 60-W tungsten bulb 2 cm from the outer glass plate (see Note 15). Allow 10–20 min for polymerization. (The polyacrylamide:BAC gel is stable for up to 72 h at 4°C.)

3.4.3. Formation and Isolation of RNAP–Promoter Complexes 3.4.3.1. FORMATION AND ISOLATION OF RNAP–PROMOTER INTERMEDIATE COMPLEX (ALL STEPS CARRIED OUT UNDER SUBDUED LIGHTING [SEE NOTE 4]) 1. Place polyacrylamide:BAC slab gel in electrophoresis apparatus and pour 0.5X TBE into upper and lower reservoirs. 2. Prerun gel for 2 h at 20 V/cm. 3. Prechill electrophoresis unit by placing in 15°C cabinet for 3 h. 4. During 15°C prechilling of step 3, dilute RNAP or RNAP derivative to 180 µg/mL (400 nM) in buffer F containing 1 mM β-mercaptoethanol and 50% glycerol, and place tubes containing diluted RNAP, derivatized DNA fragment, 2X DTT-free transcription buffer, and water, at 15°C. 5. Immediately after 15°C gel prechilling of step 3, add the following, in order, to a 1.5-mL siliconized polypropylene microcentrifuge tube: 2 µL of 5 nM derivatized DNA fragment (approx 5000 Ci/mmol), 5 µL 2X DTT-free transcription buffer, 2 µL water, and 1 µL of 180 µg/mL (400 nM) RNAP or RNAP derivative (all at 15°C). 6. Incubate 20 min at 15°C in the dark. 7. During incubation of step 6, apply voltage to gel in a 15°C cabinet: 16 V/cm. Wash wells of gel carefully with 0.5X TBE to remove unpolymerized acrylamide and BAC. (Caution: care must be exercised to avoid electrocution.)


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8. After completing incubation of step 6, immediately add 1 µL of 0.22 mg/mL heparin (prechilled to 15°C), mix, and immediately apply sample to gel in 15°C cabinet (see Note 16). Load 5 µL nondenaturing loading buffer into adjacent lane. (Caution: care must be exercised to avoid electrocution.) Continue electrophoresis in a 15°C cabinet for 20 min at 16 V/cm. Monitor gel temperature at 5-min intervals by inserting the thermocouple probe into the gel for 5 s (and removing immediately thereafter). Maintain the gel temperature at 15°C. If the gel temperature rises above 15°C, temporarily reduce electrophoresis voltage to 10 V/cm. 9. Immediately proceed to the next step (Subheading 3.4.4.).

3.4.3.2. FORMATION AND ISOLATION OF RNAP–PROMOTER OPEN COMPLEX (ALL STEPS CARRIED OUT UNDER SUBDUED LIGHTING [SEE NOTE 4]) 1. Place polyacrylamide:BAC slab gel in electrophoresis apparatus, clip 10 × 25-cm silicone heating mat directly to outer glass plate of the slab gel assembly with four large binder clips, and pour 0.5X TBE buffer in upper and lower reservoirs. 2. Prerun gel for 2 h at 20 V/cm. 3. Prewarm electrophoresis unit by placing in 37°C cabinet for 3 h. 4. During 37°C prewarming of step 3, dilute RNAP or RNAP derivative to 180 µg/mL (400 nM) in buffer F containing 1 mM β-mercaptoethanol and 50% glycerol. 5. Immediately after 37°C prewarming of step 3, add the following, in order, to a 1.5-mL siliconized polypropylene microcentrifuge tube: 2 µL of 5 nM derivatized DNA fragment (approx 5000 Ci/mmol), 5 µL of 2X DTT-free transcription buffer, 2 µL water, and 1 µL of 180 µg/mL (400 nM) RNAP or RNAP derivative (all at room temperature). 6. Incubate for 20 min at 37°C in the dark. 7. During incubation of step 6 apply voltage to the gel: 16 V/cm. Wash wells of gel carefully with 0.5X TBE to remove unpolymerized acrylamide and BAC. (Caution: Care must be exercised to avoid electrocution.) Connect heating mat to variable-voltage controller. Monitor the gel temperature at 5-min intervals by inserting thermocouple probe into the gel for 5 s (and removing immediately thereafter). Maintain gel temperature at 37°C, adjusting heater voltage as necessary (typically 12–14V). 8. After completing incubation of step 6, immediately add 1 µL of 0.22 mg/mL heparin (prewarmed to 37°C), mix, and immediately apply sample to gel (see Note 16). Load 5 µL nondenaturing loading buffer into adjacent lane. (Caution: care must be exercised to avoid electrocution.) Continue electrophoresis 20 min at 16 V/cm. Monitor the gel temperature at 5-min intervals by inserting the thermocouple probe into the gel for 5 s (and removing immediately thereafter). Maintain the gel temperature at 37°C, adjusting heater voltage as necessary (typically 12–14V). 9. Immediately proceed to next step (Subheading 3.4.4.).

3.4.4. In-Gel UV Irradiation of RNAP–Promoter Complex 1. Remove gel with both glass plates in place (see Note 17) and mount vertically in a Rayonet RPR-100 photochemical reactor equipped with 16 RPR-3500 Å tubes.

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2. Immediately UV irradiate for 3 min (17 mJ/mm2 at 350 nm) (see Note 18). 3. Immediately proceed to the next step (Subheading 3.4.5.).

3.4.5. Identification, Excision, and Solubilization of Portion of Gel Containing RNAP–Promoter Complex 1. Remove one glass plate, and cover gel with plastic wrap (leaving the other glass plate in place). Attach two autorad markers. Expose to X-ray film for 1.5 h at room temperature (see Note 19). Process the film. 2. Cut out the portion of film corresponding to the RNAP–promoter complex of interest. Using a light box, superimpose the cut-out film on gel, using autorad markers as reference points. Using disposable scalpel, excise the portion of gel corresponding to the RNAP–promoter complex. Transfer excised gel slice to a 1.5-mL siliconized microcentrifuge tube. 3. Solubilize the gel slice by adding 10 µL of 1 M DTT (approx 0.4 M final) and heating for 5 min at 37°C (see Note 20). 4. Immediately proceed to the next step (Subheading 3.4.6.).

3.4.6. Nuclease Digestion 1. During X-ray film exposure of step 1 of Subheading 3.4.5., dilute DNase I and micrococcal nuclease with ice cold nuclease dilution solution to a final concentration of 10 U/µL. 2. Transfer 10 µL of the solubilized gel slice (Subheading 3.4.5., step 3) to a new 1.5-mL siliconized polypropylene microcentrifuge tube and add 1 µL of 200 mM CaCl2, 1 µL of 0.2 mM PMSF, 0.5 µL (5 U) micrococcal nuclease, and 0.5 µL (5 U) DNase I. Incubate for 20 min at 37°C. Terminate reaction by adding 3 µL of 5X SDS loading buffer and heating for 5 min at 100°C. 3. Immediately proceed to next step (Subheading 3.4.7.).

3.4.7. Analysis 1. Apply the entire sample (16 µL) to a 4–15% gradient polyacrylamide (37.5:1 acrylamide:bis-acrylamide) slab gel. As a marker, load 5 µL prestained protein molecular-weight markers into the adjacent lane. Electrophorese in SDS running buffer at 25 V/cm until the bromophenol blue reaches the bottom of the gel. 2. Dry gel, and autoradiograph or phosphorimage.

4. Notes 1. M13mp2(ICAP-UV5) carries the lacP(ICAP-UV5) promoter, a derivative of the lacP promoter having a consensus DNA site for CAP (37) and a consensus –10 element (38). M13mp2(ICAP-UV5) was prepared from M13mp2-lacP1(ICAP) (39) by use of site-directed mutagenesis to introduce a consensus –10 element (40). M13mp2(ICAP-UV5)-rev carries the lacP(ICAP-UV5) promoter in an orientation opposite to that in M13mp2(ICAP-UV5). M13mp2(ICAP-UV5)-rev was prepared from M13mp2(ICAP-UV5) by excising the PvuII-PvuII segment corre-


2.

3. 4.

5.

6.

7. 8.

9.

10.

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sponding to positions –217 to -125 of lacP(ICAP-UV5) and inverting the PvuII– PvuII segment corresponding to positions –124 to +145 of lacP(ICAP-UV5). M13mp2(ICAP-UV5) and M13mp2(ICAP-UV5)-rev ssDNAs carry respectively the nontemplate and template strands of lacP(ICAP-UV5). M13mp2(ICAP-UV5) and M13mp2(ICAP-UV5)-rev ssDNAs are prepared as in ref. 7. The specified 10X digestion buffer is for PvuII and HaeIII. Use 10X digestion buffer recommended by supplier—omitting DTT (see ref. 41)—for other restriction enzymes. Fluorescent light and daylight must be excluded. Low to moderate levels of incandescent light (e.g., from a single bench lamp with a 60-W tungsten bulb) are acceptable. The derivatized oligodeoxyribonucleotide tolerates exposure to the Hitachi L-3000 diode-array HPLC UV detector. The derivatized oligodeoxyribonucleotide can be identified unambiguously by monitoring the UV-absorbance spectrum from 200 nm to 350 nm. The derivatized oligodeoxyribonucleotide exhibits an absorbance peak at 260 nm, attributable to DNA, and a shoulder at 300–310 nm, attributable to the azidophenacyl group. The derivatization procedure yields two diastereomers in an approximately oneto-one ratio: one in which azidophenacyl is incorporated at the sulfur atom corresponding to the phosphate O1P, and one in which azidophenacyl is incorporated at the sulfur atom corresponding to the phosphate O2P (see refs. 4 and 6). Depending on oligodeoxyribonucleotide sequence and HPLC conditions, the two diastereomers may elute as a single peak, or as two peaks (e.g., at 24% and 25% solution B). In most cases, no effort is made to resolve the two diastereomers, and experiments are performed using the unresolved diastereomeric mixture. This permits simultaneous probing of protein–DNA interactions in the DNA minor groove (probed by the O1P-derivatized diastereomer) and the DNA major groove (probed by the O2P-derivatized diastereomer). Phenyl azides are unstable at temperatures above 70°C. Avoid heating above 70°C. HaeIII digestion, which yields a DNA fragment corresponding to positions –141 to +63 of lacP(ICAP-UV5), is used for preparation of DNA fragments derivatized between positions –80 and –1, inclusive. PvuII digestion, which yields a DNA fragment corresponding to positions –124 to +145 of lacP(ICAP-UV5), is used for preparation of DNA fragments derivatized between positions +1 and +80 inclusive. (Use of DNA fragments with >60 bp between the site of derivatization and the nearest DNA fragment end eliminates “nonspecific” crosslinking from the subpopulation of complexes having RNAP bound at a DNA-fragment end [42] rather than at the promoter.) Pour 10 mL Ni:NTA–agarose suspension into a 20-mL Econo-Pac column. Remove snap-off tip at bottom and allow liquid to drain. Place the frit on the top of the column bed. BAC is a disulfide-containing analog of bis-acrylamide (43–45) Polyacrylamide:BAC gels can be solubilized by addition of reducing agents (43–45). The synthesis of BAC in this chapter is adapted from ref. 43.

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11. Acryloyl chloride reacts violently with water. Add acryloyl chloride in 0.5-mL portions, waiting 30 s between successive additions. 12. BAC is substituted for bis-acrylamide on a mole-equivalent, not mass-equivalent, basis (43–45). The solubility of BAC in water is increased by adding acrylamide before adding BAC and by performing additions at 60°C. 13. TEMED and ammonium persulfate concentrations are critical variables in the preparation of polyacrylamide:BAC gels (44,45). (Use of nonoptimal TEMED and ammonium persulfate concentrations in preparing of polyacrylamide:BAC results in difficulties in subsequently solubilizing gels.) 14. Siliconize notched glass plate by applying 30 µL SurfaSil siliconizing agent and spreading evenly with a Kimwipe. 15. Heating during polymerization yields polyacrylamide:BAC gels that are maximally solubilizable upon the addition of reducing agents (44,45). Heat the glass plates of the gel assembly evenly. (If necessary, use two task lamps.) Avoid heating above 70°C, as this can result in the formation of bubbles and/ or detachment of gels from the glass plates. 16. Do not add loading buffer to the reaction mixture. The reaction mixture is sufficiently dense for loading (because of the presence of glycerol). 17. Ultraviolet irradiation is performed with both glass plates in place. The glass plates exclude wavelengths [D]t and (b) [D]t >> KD (see Note 2). • When a wild type restriction endonuclease is used to cut its natural DNA target, both strands of the duplex are usually cut at the same rate. However, introducing a modification into only one of the DNA strands often results in the individual strands being cut at different rates. In these cases cleavage is most simply and accurately measured if the two substrate strands and the two labeled product strands can be separated, as shown in Fig. 1. • Measurement of DNA cleavage by restriction endonucleases, under single-turnover conditions, approaches the limit of manual manipulation. In some cases, it has been possible to mix the enzyme with its substrate and withdraw aliquots by *Throughout this chapter, E = endonuclease; D = DNA; ED = enzyme–DNA complex. The subscripts t and f denote the total and free amounts of E and D present (i.e., [E]f = [E]t – [ED]; [D]f = [D]t – [ED]). Several investigators use KA (= 1/KD) rather then KD. Both terms are acceptable, and in some instances in this chapter KA has been used.

Assay of Restriction Endonucleases

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Fig. 1. Oligonucleotides that have been used to measure single turnovers with the EcoRV and EcoRI restriction endonucleases (recognition sequence shown with line, cleavage sites with arrows). With EcoRV, both the substrate strands are different lengths. In the case of EcoRI, the two substrate strands can be separated by denaturing gel electrophoresis, despite having the same length, as the top strand is dG rich and the bottom dC rich. dC-rich strands migrate faster than dG (dA and T base-pairs affect migration much less). With EcoRV 3' labeling (*ddA) was used, and with EcoRI, 5' labeling (*P) was used. The offset recognition sites gives products of different sizes. The two arrangements ensure the separation of both substrate and both labelled product strands and so allow independent measure of the rate of cleavage of the two strands. Only the labeled products are shown. For both enzymes the “top” strands are written in the 5' → 3' direction and the “bottom” are written in the 3' → 5' direction. hand. In others, the reaction is too fast to be evaluated in this manner and a rapidmixing quenched flow apparatus must be used. Alteration to the endonuclease or the DNA often cause a reduction in the single-turnover rate constant and it is often possible to use manual methods in these cases.

The most suitable method for KD determination depends critically on its magnitude and hence the stability of the protein–DNA complex (see Note 3). Several approaches for the measurement of KD are presented, enabling equilibrium constant evaluation under conditions that range from very tight (KD ≈ pM) to very weak (KD ≈ mM) binding. When commencing experiments, the KD will not be known and good practice requires an initial estimate, followed by an accurate evaluation using the optimal approach. Modification to the endonuclease or the DNA often weakens their interaction, sometimes by a considerable amount. Therefore, the method used in the “natural” case may not be the best when alterations to the macromolecules are present. Whatever method is used, it is important to prevent DNA hydrolysis and this is most simply achieved by omission of Mg2+ and addition of EDTA. Two commonly used methods for KD determination are filter binding and gel retardation (electrophoretic mobility shift assay [EMSA]). Both are discussed elsewhere in this volume (see Chapters 1 and 2) and this chapter

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emphasizes their application to restriction endonucleases and also the use of competition titration. Most often, a 32P-labeled oligonucleotide is incubated with increasing amounts of restriction enzyme to produce a protein–DNA complex. The free and protein-bound DNA are then separated and the relative amounts in each pool determined. This allows the construction of a binding isotherm and KD evaluation. Data analysis is simplified when [E]t ≈ [E]f and experimentation is usually carried out under these conditions (see Note 4). It is also useful to carry out a “reverse” titration: incubating a fixed amount of endonuclease with increasing quantities of 32P-labeled oligonucleotide. Titrations with a fixed [DNA] may give rise to shifts in the oligomeric state of the protein because of its increasing concentration, which can produce biphasic isotherms or curves that do not show asymptotic behavour. This arises due to the coupled reactions E2 + E2 → E4 → En (where E2 is the active form) and is particularly relevant to modified substrates that show weak binding; requiring titration with concentrations of protein that may exceed the KD for the coupled dimer–tetramer equilibrium. If both procedures give identical KD values, one has confidence that the coupled reaction can be ruled out as an obfuscating factor. In addition, the combination of the two approaches permits precise determination of the number of active protein molecules, providing that the DNA concentration is accurately known. Filter binding and gel retardation differ in the manner used to separate the free DNA and the protein–DNA complex. In filter binding, separation is achieved using a nitrocellulose filter that retains proteins, and hence protein– DNA complexes, but allows the passage of free DNA. Some proteins are poorly bound by nitrocellulose filters although this difficulty can sometimes be circumvented by using a different filter material (e.g., pure nitrocellulose rather than mixed-ester filters). Gel retardation uses non denaturing PAGE; free DNA migrates more rapidly than the protein–DNA complex giving, under ideal conditions, two well-resolved bands. Both approaches are sensitive (can be used at very low [DNA] to measure tight binding), experimentally very simple to carry out, and do not require specialized equipment. Whether filter-binding or gel-shift methods are chosen, the direct titration protocol is not recommended for modified substrates that show very weak binding (i.e., KD > 10–7 M), because the lifetime of the complexes (see Note 3) is always shorter than the time required either for filtration or for entry into the gel (see Note 5). This may result in partial dissociation of the endonuclease– DNA complex during the measurement (i.e., a “nonequilibrium” situation). For weak binding, a competition titration (6) protocol should be used. Here, a fixed quantity of an endonuclease and a 32P-labeled oligonucleotide (most often containing an unmodified recognition sequence for the endonuclease under study) are allowed to form a complex. Progressively increasing amounts of a


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Fig. 2. The structure of hexachlorofluorescein and its linkage to the 5'-phosphate of an oligonucleotide.

nonradioactive competitor DNA (e.g., a variant oligonucleotide in which there has been a base-analog or natural base-pair substitution) are added. The unlabeled DNA molecules compete with the 32P-labeled oligonucleotide for the DNA-binding site on the protein and so leads to a displacement of the radioactive probe. It is good practice to compare some of the KD values obtained by the direct method with those obtained by competition titration, both to test whether equilibrium is significantly perturbed in the direct method and to provide confidence that all binding reactions have achieved equilibrium in the more indirect competition method. Thus, one should include in the experimental set of modified oligonucleotides at least one “internal control” for which both direct and competition titrations can be performed (see Note 6). Equilibrium constants can also be determined using fluorescence anisotropy (7,8) with oligonucleotides labeled with hexachlorofluorescein. When a fluorophore is excited with plane polarized light, the extent to which the emitted light becomes depolarized depends on the rate at which the fluorophore tumbles (which, in turn, depends on molecular weight) and also the lifetime of the fluorescence excited state (see Note 7). When a protein binds to an fluorescent labeled oligonucleotide, the mass associated with the fluorophore, and therefore the fluorescence anisotropy, is increased, allowing KD determination. Hexachlorofluorescein is commercially available as a phosphoramidite suitable for automated DNA synthesis, enabling its attachment to the 5' terminus of an oligonucleotide via a six-carbon-chain linker (Fig. 2). The flexible linker gives the probe a degree of motion independent of the dynamics of the DNA or the protein–DNA complex. However, although the probe is not rigidly attached to the DNA, it has enough movement coupled to the DNA, together with an appropriate fluorescence lifetime of about 3 ns for the probe attached to a 21-mer (9), to make it sensitive to protein binding (see Note 7). The minimum concentration of hexachlorofluorescein that can be easily measured is about 1 nM and this limits the technique to measuring KD values of approx 1 nM and above. It has been used to measure KD values that approach

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1 µM. Thus, fluorescence anisotropy is much less sensitive than filter binding or gel retardation, which rely on the detection of 32P. The biggest advantage of this method is that it is a strictly equilibrium approach and does not suffer from problems caused by protein–DNA dissociation. Drawbacks include the requirement for expensive equipment and inability to measure tight binding (KD < 1 nM). 2. Materials

2.1. Oligodeoxynucleotides and Restriction Endonucleases 1. [32P]-labeled oligodeoxynucleotides. These should be of high specific activity, prepared using either γ-[32P]-ATP (3000–6000 Ci/mmol)/polynucleotide kinase (5' labeling) or α-[32P]-ddATP(>5000 Ci/mmol)/terminal transferase (3'-labeling) and purified by standard procedures (10). Duplex oligodeoxynucleotides are prepared by heating equimolar amounts of each strand to 95°C in buffer with the same pH value and salt concentration to be used in kst or KD measurement and cooling slowly to room temperature. For kst determination, both strands must be labeled; for KD evaluation, only one strand needs to be labeled. 2. Oligodeoxynucleotides containing hexachlorofluorescein at their 5' termini. These can be prepared by chemical synthesis using commercially available hexachlorofluorescein–phosphoramidite (Glen Research, Sterling, VA; Cruachem Ltd., Glasgow, Scotland) and purified by reverse-phase high-performance liquid chromatography (HPLC) (9). Duplexes, only one strand of which needs to contain the fluorophore, are prepared as in item 1. 3. Restriction endonucleases under study. In this case, EcoRI, EcoRV, and BamHI purified from overproducing Escherichia coli strains.

2.2. kst Determination 1. Vertical slab gel apparatus (140 × 160 k 0.75 mm) (e.g., Hoefer SE600 or equivalent) and power pack with ≈200 V direct current output. 2. Denaturing polyacrylamide gel. Prepared from 16% acrylamide (acrylamide/bisacrylamide, 19/1) in 0.089 M Tris–borate, pH 8.0, containing 8 M urea and 1 mM EDTA and polymerized with 0.05% ammonium persulfate (added from a freshly prepared 10% aqueous solution) and 1% TEMED (N,N,N'N'-tetramethylethylene diammine). The gel should be prerun at a constant power of 30 W, for 1 h, prior to use. 3. Gel running buffer: 0.089 M Tris–borate, pH 8.0, containing 1 mM EDTA. 4. Hydrolysis buffer (made up at 2X the final concentration, freshly prepared and stored at 4°C): in the examples given using EcoRV endonuclease; 20 mM HEPES, pH 7.5, 200 mM NaCl, and 20 mM MgCl2 (see Note 8). 5. Enzyme dilution buffer: Incubation buffer plus 5% (v/v) glycerol (see Note 8). 6. Stop solution: 0.1 M Tris-HCl, pH 8.0, 0.1 M EDTA, 2.5 M urea, 10% (w/v) sucrose, and 125 mg/mL (each) bromophenol blue and xylene cyanol FF. 7. Vacuum bag sealer and plastic sheets. 8. Phosphorimager (e.g., Fuji BAS-150) and phosphorimager screen (e.g., Fuji BAS-MP) (or equivalent).


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9. Quenched flow apparatus (e.g., Hi-Tech RQF–63 [Hi-Tech Scientific, Salisbury, UK]) or equivalent.

2.3. KD Determination (Filter Binding) 1. Vacuum filtration manifold (e.g., Millipore 1225 Sampling Vacuum Manifold, Bedford, MA). 2. Mixed ester nitrocellulose membrane filters (e.g., Gelman GN6, Millipore HAWP02500, Schleicher & Schuell ME25) or pure nitrocellulose membrane filters (e.g., Schleicher & Schuell, type BA85), 25 mm disks, 0.45-µm pore size. 3. Enzyme dilution buffer: Binding buffer plus 5% glycerol and 0.2 M or 0.6 M NaCl (final concentrations), pH 7.4 (see Note 8). 4. Filter buffer (stored at 4°C): 10 mM BTP (bis-Tris propane), 1 mM EDTA, 0.02% NaN3 at desired pH (e.g., pH 7.4) and desired salt concentration (e.g., 0.2 M NaCl) (see Note 8). 5. Binding buffer (stored at 4°C): Filter buffer plus 0.1 mg/mL bovine serum albumin and 50 µM dithiothreitol (DTT). 6. Polyethylene liquid-scintillation vials (6 mL capacity). 7. Liquid-scintillation fluid (e.g., Scintisafe 30%, Fisher Biotech, Pittsburgh, PA). 8. Liquid-scintillation counter (e.g., Packard model 1600; Meridan, CT).

2.4. KD Determination (Gel Retardation) 1. Vertical slab gel apparatus (140 × 160 × 0.75 mm) (e.g., Hoefer SE600 or equivalent) and power pack with ≈200 V direct current output. 2. 10% Nondenaturing polyacrylamide gel (acrylamide/bis-acrylamide, 37.5/1) in 0.089 M Tris–borate, pH 8.0, and 1 mM EDTA and polymerized with 0.05% ammonium persulfate (added from a freshly prepared 10% aqueous solution) and 1% TEMED. The gel should be prerun at a constant power of 20 W with gel running buffer and the wells rinsed with this buffer, prior to use. 3. Gel running buffer: Usually 0.089 M Tris–borate, pH 8.0, containing 1 mM EDTA. However, the running buffer can, to a limited extent, be varied to produce a better match with the binding buffer (see Note 9). 4. Binding buffers and enzyme dilution buffers: As in Subheading 2.3., items 3 and 5 except that the binding buffer should additionally contain 3% (v/v) glycerol (see Note 10). 5. Bromophenol blue solution: 0.25% (w/v) in 30% (v/v) glycerol. 6. Vacuum bag sealer and plastic sheets. 7. Phosphorimager e.g., Fuji BAS–150 and phosphorimager screen (e.g., Fuji BAS-MP) (or equivalent).

2.5. KD Determination (Fluorescence Anisotropy) 1. Fluorimeter, with thermostated cuvet compartment, capable of measuring fluorescence anisotropy e.g., Aminco SLM-8100. 2. 3-mm-thick 570-nm longpass filter, (OG-570, Schott Glaswerke). 3. 0.5 mL semimicro quartz fluorescence cuvets (excitation and emission pathlengths 5 mm).

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4. Fluorescence binding buffer and enzyme dilution buffer: In the examples given with the EcoRV endonuclease, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.1 mg/mL acetylated bovine serum albumin. The fluorescence binding buffer should be prepared using the best quality reagents and HPLC-grade water and both degassed and passed through 0.22-µm filters (e.g., Millex-GV13, Millipore) prior to use.

2.6. Data Analysis Software capable of fitting reaction data to single and multiple exponential decay(s) and binding data to single-site binding isotherms using nonlinear regression analysis. The software used for binding analysis should be able to deal with direct titrations when the simplifying assumption [E]f = [E]t cannot be made (see Note 4) and competitive titrations. Three packages described in this chapter are GraFit (11), Scientist (12), and SigmaPlot (13), but any software with a similar capability is perfectly acceptable. 3. Methods 3.1. Determination of kst

3.1.1. Slow Hydrolysis (t1/2 15 s or More ≈kst Values Slower Than 3 min–1; KD Between 2 and 40 nM) (e.g., with Mutant EcoRV or Altered Oligodeoxynucleotides) 1. In a plastic microcentrifuge tube, prepare 11 µL of a solution containing 1.5 µM of radiolabeled DNA duplex in hydrolysis buffer (in this case for the EcoRV endonuclease, 10 mM HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl2) (see Note 8). Keep on ice. 2. Withdraw 1 µL of the above solution and add to 10 µL of stop solution (item 6 of Subheading 2.2.). This serves as a zero time-point. 3. Prepare 5 µL of a solution containing 30 µM of EcoRV endonuclease in 10 mM HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl2. Keep on ice. 4. Incubate the solutions prepared in steps 1 and 3 at 25°C for 10 min. 5. Mix the two solutions to give a final oligonucleotide concentration of 1 µM and a final endonuclease concentration of 10 µM (see Notes 1 and 2). Vortex briefly to ensure thorough mixing and incubate at 25°C. 6. Withdraw 1-µL aliquots at times up to 25 h (the exact time range to be used must be found by trial and error and will depend on the particular mutant enzyme and/or altered oligodeoxynucleotide under study) and quench the reaction by the addition to 10 µL of stop solution (item 6 of Subheading 2.2.). Store the quenched samples on ice. 7. Run the samples on 16% denaturing polyacrylamide gels (item 2 of Subheading 2.2.) until the bromophenol blue dye marker reaches the bottom of the gel. 8. Remove the gel from the electrophoresis apparatus and seal in a plastic bag using a vacuum bag sealer.


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9. Determine the amount of radioactivity present in each substrate and product band using a phosphorimager (see Note 11). 10. From the data obtained from the phosphorimager, determine the percentage of the two substrate and the two product strands present at each time-point.

3.1.2. Rapid Hydrolysis (t1/2 15 s or Less ≈kst Values Faster Than 3 min–1; KD Between 2 and 40 nM) (e.g., with Wild-Type EcoRV and Cognate GATATC Recognition Sites 1. Fill the three syringes of the quenched flow apparatus with the following: a. Reaction syringe 1: 0.1 mL of 2 µM radiolabeled DNA in 10 mM HEPES, pH 7.5, 100 mM NaCl and 10 mM MgCl2. b. Reaction syringe 2; 0.1 mL of 20 µM EcoRV endonuclease in 10 mM HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl2. c. Quench syringe 3; 0.1 mL of 0.3 M EDTA. 2. Set the apparatus to mix the 0.1 mL of the oligonucleotide and enzyme solution (final concentrations of each 1 µM and 10 µM, respectively) and to quench the reaction at the first time-point (0.051 s) by the addition of the 0.1 mL of the EDTA solution. 3. Keep the quenched sample on ice. 4. Repeat for each subsequent time-point (in this case, 19 further points between 0.094 and 20 s) (see Fig. 4). 5. As a zero time-point, 0.1 mL of the oligonucleotide solution manually mixed with 0.1 mL of 10 mM HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl2, and 0.1 mL of 0.3 M EDTA can be used. 6. Add 5 µL of each of the quenched samples to 5 µL of stop solution (Subheading 2.2., item 6). 7. Proceed with the analysis by gel electrophoresis and phosphoroimaging (Subheading 3.1.1., step 6–9).

3.1.3. Data Analysis; kst Determination 1. Cleavage of a duplex oligonucleotide by a restriction endonuclease involves parallel sequential reactions (Fig. 3), giving intermediates in which one strand is nicked, and described by four rate constants (14,15). In some instances, the reaction scheme can be simplified. This is usually true for wild-type endonucleases acting on their natural, unmodified, target sequences. Here, both strands are efficiently cut in a concerted reaction and nicked intermediates do not accumulate. In this case, the cutting of each of the two substrate strands (or the accumulation of the two radiolabeled products) can be described by a single rate constant with an acceptable degree of accuracy. 2. If simplification is possible, fit the data to an equation describing a single exponential using GraFit (11). This equation is supplied within GraFit and almost all biological kinetic software packages. The design of the oligonucleotides used (Fig. 1) means that the cutting of each strand can be evaluated individually. GraFit requires time and the percentage of substrate or product present at these times to

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Fig. 3. Parallel sequential cutting of a double-stranded oligodeoxynucleotide by the EcoRV endonuclease. be entered into a spreadsheet and to use these data to evaluate kst. Concerted cutting of both strands implies that the kst for each of them should be, within the limits of experimental error, identical. An example is shown in Fig. 4. 3. With modified oligonucleotides or enzymes altered by mutagenesis, cutting often becomes inefficient and nicked intermediates accumulate. Altering any one point in a palindromic recognition site produces structural asymmetry, leading to different values of k1 and k2. In such cases, the following equations are used to obtain values of the four rate constants that describe the unsimplified reaction scheme (Fig. 3): k2 P1 = S1[1 – eλt – — (e–λt – ek4t)] k4 – λ k1 P2 = S2[1 – eλt – — (e–λt – ek3t)] k3 – λ where λ = k1 + k2 and S1 and S2 are the percentages of total radioactivity in each DNA substrate strand at t = 0. If end labeling of the two strands is equal, then S1 = S2 = 50%. The ability to separate the two DNA substrate strands permits normalizing for “differential labeling” in the event that the two strands have been radiolabeled with slightly different efficiencies (e.g., if S1 = 40% and S2 = 60%, these values should be entered into the equations). If the radioactivity of the two strands is markedly different, the program has more difficulty finding the best fit. This is especially the case when a strand that has an intrinsically faster cleavage rate constant has lower specific radioactivity than the other strand. Therefore, it is preferable to attempt to label both strands to the same specific activity. Reaction times (t) and the amount of products (P1 and P2, expressed as percentage of total radioactivity for each time point) are entered into the Scientist (12) software spreadsheet. A plot of the experimental and “fitted” values of P1 and P2 is generated along with numerical values of the rate constants k1 to k4. Such fits are most statistically robust when the uncertainties in the independent


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Fig. 4. Cleavage of the duplex oligonucleotide (1 µM) produced by mixing 5'-AAAGTCTGTGGATATCCAAGTGGCTACCGT-*ddA and 5'-CCCCCACGGTAGCCACTTGGATATCCACAGACT-*ddA (Fig. 1) with EcoRV endonuclease (10 µM) using a quench-flow apparatus. The top part shows an autoradiograph of denaturing PAGE analysis of the two substrates and the two labeled products present after various mixing times (given on top of the gel lanes in s). The bottom part shows fits for the formation of each product using the simplified model (i.e., formation of each product is fitted using a single exponential with Grafit [11]). It should be noted that data were obtained by phosphorimaging of the gel, and the autoradiograph is presented for illustrative purposes only. The kst values found were 0.94 s–1 and 0.77 s–1 for the production of P2 and P1 respectively. Fits to the full parallel sequential model (figure 3) using Scientist (12) (not shown) gave a k2 (for P2 and production) of 0.65 s–1 and a k1 (for P1 production ) of 0.51 s–1. Thus, in the case of wild-type EcoRV with its cognate GATATC sequence, the simplified model gives rates that are very similar to the rigorously correct full model.

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Connolly et al. variable (time) are much smaller than the uncertainties in the dependent variable (here, each radioactivity measurement in a product) and when the number of data points is sufficiently high to provide a good sampling of experimental uncertainties. The “goodness of fit” of the experimental data to the fitted curve is judged by the individual reaction point residuals, which should vary no more than 10%. We have found that a rigorous test of the robustness of the fit to the above equations is to measure the second strand cleavage rate constants for “nicked” constructs, assembled from three individual fragments. When using the full parallel-sequential model, it is good practice to apply the same calculation method to reference reactions with the normal enzyme–substrate pair. A comparison of the rate constants obtained from the “full” and “simplified” calculation methods then tests the validity of using the simplified-model fit for the cleavage rate constants k1 and k2 for the reference pair (see Note 12).

3.2. Filter Binding 3.2.1. K D Determination: Direct Binding–Titration of DNA with Protein. Example: KD = 0.66 nM KA = 1/KD = 1.5 × 109 M–1 1. Presoak nitrocellulose membrane filters in filter buffer at the same salt concentration and pH used in the binding reaction. Place in the vacuum manifold. 2. Prepare a solution of 0.5 nM radiolabeled duplex DNA by diluting the radiolabeled stock DNA solution (100 nM) with binding buffer at the desired salt concentration and pH. Add 10 µL of radiolabeled duplex oligonucleotide (0.5 nM) to each of 10 microcentrifuge tubes; the final concentration of radiolabeled duplex DNA should be 0.05 nM after step 3 (see Note 4). Add the appropriate volume of binding buffer such that the total volume of the reaction after step 3 would be 100 µL. Equilibrate on ice for 5 min. 3. To each of the above microcentrifuge tubes add restriction endonuclease to give final concentrations ranging from 0.05 nM to 5 nM (i.e., the midpoint protein concentration should be approx equal to the KD; see Note 4); equilibrate reactions on ice for 5 min more. During an experimental series, the protein stock should always be kept on ice and any intermediate dilutions of the stock that are not used directly in the final reactions are made using enzyme dilution buffer (salt type and pH always match the experimental conditions). The final dilution to give the sample used in the reaction should be made with binding buffer (no glycerol) at the appropriate salt concentration (see Note 8). The salt derived from the diluted enzyme stock and the DNA source is always accounted for in designing the experiment. 4. Set up two “blank” tubes containing DNA, binding buffer, but no enzyme. One blank tube will be filtered to obtain RB (background counts) and the other used to determine RT (the input counts). 5. Also set up a tube containing DNA, binding buffer, and enzyme at a concentration 100-fold the KD (here, 50 nM enzyme). This tube will be filtered to obtain Rmax, which represents the total available DNA for binding.


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6. Transfer the reaction tubes, the two blank tube, and the Rmax tubes to the desired temperature (e.g., 25°C) and incubate for 30 min. 7. Pipet 85 µL from each reaction tube, the RB blank, and the Rmax tube onto a presoaked 25 mm nitrocellulose filter in the vacuum filtration manifold with the vacuum applied. The vacuum (flow rate about 0.5 mL/10 s) is applied continuously throughout the experiment to pull the reaction aliquot through the filter. 8. As quickly as possible, wash each filter with 350 µL of filter buffer to remove trapped free DNA. 9. Place each filter in a liquid-scintillation vial and add 2.5 mL of liquid-scintillation fluid. Count in a liquid scintillation counter. 10. Pipet 85 µL from the RT blank tube onto a nitrocellulose filter. Do not wash, but place the filter directly into a liquid-scintillation vial, add 2.5 mL of liquid-scintillation fluid, and count.

3.2.2. KD Determination: Direct Binding–Titration of Protein with DNA Example: KD = 40 pM, KA = 1/KD= 2.5 × 1010 M–1 1. Presoak nitrocellulose membrane filters (Subheading 3.2.1., step 1). 2. Prepare a 4 nM stock solution of radiolabeled DNA. For a 4 nM stock DNA solution, the ratio of radiolabeled DNA to unlabeled DNA is typically 1:3 or 1:4, depending on the specific activity of the radiolabeled DNA. 3. To each of 10 microcentrifuge tubes, add radiolabeled DNA to give final concentrations ranging from 10 pM to 600 pM. (the midpoint DNA concentration should be approx equal to the KD, see Note 4). 4. Add the appropriate volume of binding buffer plus salt at the desired pH such that the total volume of the reaction after step 6 will be 100 µL. Transfer to ice and equilibrate for 5 min. 5. Prepare a solution (40 pM) of restriction endonuclease, at the desired salt concentration and pH, by making intermediate dilutions of the stock enzyme solution with enzyme dilution buffer and the final dilution (to give the 40 pM solution) with binding buffer as described in Subheading 3.2.1., step 3. 6. To the 10 reaction tubes prepared above, add 10 µL enzyme to give a final concentration of 4 pM. Keep on ice for an additional 5 min. 7. Set up 10 “blank” reaction tubes (each with total volume 100 µL) that correspond to the reaction tubes prepared in steps 3 and 4 (i.e., 100 µL total volume; radiolabeled DNA concentrations ranging from 10 pM to 600 pM) except that no enzyme is added. 8. Transfer the reaction and blank tubes to the desired temperature (e.g., 25°C) and incubate for 30 min. 9. Remove 85 µL from each tube and carry out filter binding as described in Subheading 3.2.1., steps 5–7. As the DNA concentration varies in this experiment, a different blank (with a DNA concentration that corresponds to its reaction partner) is required for each reaction point.

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3.2.3. Data Analysis: Direct Binding 1. For “normal titrations,” where DNA is titrated with protein, values of Kobs are obtained by nonlinear least-squares fits to a single-site binding isotherm: [ED] KA[E] f = [D]t 1 + KA[E]f Note that here Kobs is KA and not KD (see the footnote in Subheading 1.). The actual equation entered into SigmaPlot is (this titration is carried out under conditions [E]f ≈ [E]t, see Note 4): RF – RB = MAX

KA[E] t 1 + KA[E]t

The experimental data entered into the spreadsheet are the enzyme concentration, [E]t, for each titration point and RF (counts representing complex retained on the filter) for each titration point; RB (counts obtained when radiolabeled DNA is filtered without enzyme). A plot of the experimental counts (circles) and idealized counts (line) versus [E]t is generated as shown in Fig. 5. In this figure, [ED]/ [ED]max i.e., (RF – RB)/MAX is plotted against [E]t. The nonlinear least squares best fit to the equation will give both KA and MAX. The parameter MAX is the asymptote of the binding isotherm and represents the theoretical maximum counts retained by the filter. This theoretical MAX can be checked against the experimentally determined Rmax (see Subheading 3.2.1., step 5). The retention efficiency, MAX/(RT – RB) or Rmax/(RT – RB), is a good index of reproducibility for each set of conditions (enzyme, DNA, salt, pH, temperature). The three enzymes (EcoRI, BamHI, and EcoRV) have different retention efficiencies. 2. In the case of “reverse titration” (i.e., when protein is titrated with DNA), values for KA are obtained essentially as above, except that here the equation for a singlesite binding isotherm is

(

[ED] KA[D] f KA[D]t = and RF – RB = MAX [E]t 1 + KA[D]f 1 + KA[D]t

)

Because the concentration of enzyme is held constant and titrated with DNA, [E]t should be 5–10% of the KD value (see Note 4). This is in contrast to a “normal titration” (DNA held constant and titrated with protein), where [D]t is 5–10% of the KD.

3.2.4. KD Determination: Competitive Equilibrium Binding. Examples: KD of Reference DNA = 0.25 nM; KD of Competitor DNA = 0.69 nM KD of reference DNA = 0.25 nM; KD of competitor DNA = 0.81 µM 1. Presoak nitrocellulose membrane filters as in Subheading 3.2.1., step 1. 2. To each of 10 microcentrifuge tubes, add 10 µL of radiolabeled reference specific DNA from an 10 nM stock solution to give a final concentration of 1 nM (see Note 12).


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Fig. 5. Representative binding isotherm determined by the direct equilibrium filterbinding assay. The specific BamHI substrate 5'-CGCGGGCGGCGGATCCGGGCGGGC was titrated with BamHI endonuclease. The binding buffer contained 0.14 M potassium acetate at pH 7.3 and the experiment was carried out at 25°C. Solid circles are experimental points. The fitted Sigmaplot curve gives a KA value of 1.51 × 109/M. 3. Add the appropriate volume of unlabeled competitor DNA such that the final concentrations of competitor DNA will vary between 0 and 40 nM for an expected KD of competitor DNA ≈1 nM (see Fig. 6A). If the KD expected for the competitor DNA is much higher (e.g., approx 1 µM), then the final concentrations of unlabeled competitor DNA should vary in the range from 0 to 20 µM (see Fig. 6B). Add the appropriate volume of binding buffer (at desired salt concentration and pH) such that the total volume of the reaction after step 4 will be 100 µL. Place tubes on ice for at least 10 min. 4. To each of the above 10 microcentrifuge tubes, add restriction endonuclease to give a final concentration of 0.8 nM, taking into account the considerations detailed in Subheading 3.2.1., step 3 for enzyme dilution (see Notes 8 and 12). Keep on ice for 5 min or more. 5. Set up two additional tubes: a “blank” tube containing radiolabeled DNA, binding buffer, but no competitive DNA and no enzyme to give RB and a tube to obtain the Rmax (i.e., maximum counts retainable by the filter) containing radiolabeled DNA, binding buffer, and a final concentration of 80 nM enzyme (no competitive DNA). The Rmax tube should also be chilled on ice. 6. Transfer all reactions to the desired temperature (e.g., 25°C) and equilibrate for 30 min. 7. Remove 85 µL from each tube and carry out filter binding as described in Subheading 3.2.1., steps 5–7. Background counts (RB) are obtained by filtering the tube containing radiolabeled DNA in the absence of protein and competitor DNA.

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Fig. 6. Representative equilibrium-competition curves for the interaction of BamHI endonuclease with specific and nonspecific sites. A 40-base-pair specific “snapback” substrate 5'-TGGGTGGGATCCCACCCACCCCCTGGGTGGGATTCCACCC was used as a radiolabeled probe for both competition curves. The binding buffer contained 0.14 M potassium acetate at pH 7.3 and the experiment was carried out at 25°C. Solid circles are experimental points. Test unlabeled specific competitors were (a) the same specific substrate used for the direct binding assay in Fig. 4 and (b) a nonspecific site (CCTAGG) embedded in the same flanking context. The KD value determined from the Sigmaplot fit to curve A is 0.69 nM (KA = 1.45 × 109 M–1 ) and to curve B is 0.81 mM (KA = 1.23 × 106/M–1). Corrected reaction counts are obtained by subtracting the background counts from each reaction. The tube containing radiolabeled DNA and 80 nM enzyme is used to determine the Rmax value (see step 5).


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3.2.5. Data Analysis: Competitive Equilibrium Titration Results are fitted, using SigmaPlot, to a binding isotherm as previously described (6) using the equation [ED1] =

[E] t ([D1]t – [ED1]) [D ] K1 1 + 2 t + ([D1]t – [ED1]) K2

(

)

where [E]t = total enzyme concentration, [D1]t = total radiolabeled reference DNA concentration, [D 2] t = total unlabeled competitor DNA concentration, K1 = dissociation constant for the radiolabeled reference DNA; K 2 = dissociation constant for the unlabeled competitor DNA. Solving the equation for [ED1] yields [ED1] =

1 K K K1 + 1 [D2]t + [E]t + [D1]t – (K1 + 1[D2]t + [E1]t + [D1]t)2 – 4[D1]t[E1]t 2 K2 K2

{

冪[

]}

where [ED1] + [D1]t

RF – RB Rmax – RB

The dissociation constant K1 for the reference DNA is always determined by direct equilibrium binding at the start of a competition experiment. Rmax, obtained at saturating concentrations of protein and without competitor DNA (see Subheading 3.2.4., step 5) represents the maximum available [D1]t. The known values for K1, [D1]t, [E], Rmax, and RB (see Subheading 3.2.4., step 5), are entered into the SigmaPlot software at the start of each calculation. The experimental data entered into spreadsheet columns are the corrected counts (counts retained on the filter for each titration point minus background counts; RF – RB) and the competitor DNA concentration [D2]t for each point. The curve generated by plotting the corrected counts as a function of increasing concentrations of competitor DNA ([D2]t) is fitted to the best value for the equilibrium dissociation constant K 2 using SigmaPlot nonlinear regression analysis. Figure 6 presents the results as a plot of the ratio of [ED1]/[ED1]0 where [ED1]0 is the concentration of enzyme–DNA complex obtained in the absence of competitor. The [ED1]0 found in this experiment should not be significantly different from the [ED1]0 calculated for a direct binding experiment.

3.3. Gel Retardation 3.3.1. KD Determination: Direct Binding–Titration of DNA with Protein 1. Set up and equilibrate binding reactions as described in Subheading 3.2.1., steps 2–4. The final reaction tubes should additionally contain 3% (v/v) glycerol (see Note 10). A “blank” should contain all the components except endonuclease. Smaller samples (typically 10 µL) are used in gel retardation than are used for

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2.

3.

4. 5.

Connolly et al. filter binding (85 µL in the examples given above). Therefore, if only gel retardation is being carried out, the final volume should be 10 µL. Alternatively, it can be very useful to make up a binding reaction and split into aliquots for simultaneous analysis by both filter binding and gel retardation (either direct or competition titration). Following incubation, load the aliquots of the samples, typically 10 µL, into the wells of 10% nondenaturing polyacryalmide gels (see Subheading 2.4., items 1 and 2 and Note 9). Run the gels at a constant power of 20–25 W, with cooling, until a bromophenol blue marker (see Subheading 2.4., item 5 and Note 10) in an adjacent lane, not containing the experimental samples, reaches the gel front (about 1 h). Remove the gel from the electrophoresis apparatus and seal in plastic using a vacuum bag sealer. Determine the amount of radioactivity present in the bands that correspond to the free and bound DNA using a phosphorimager (see Note 11).

3.3.2. KD Determination: Competitive Equilibrium Binding 1. Set up equilibrium competition reactions according to the steps outlined in Subheading 3.2.4., steps 2–4, except that the binding buffer for all reactions should include 3% (v/v) glycerol (see Note 10). If only gel retardation is being carried out smaller volumes (see Subheading 3.3.1., step 1) (e.g., 10 µL should be used. A “blank” should contain all the components except endonuclease and competitor DNA. 2. Perform gel retardation analysis and phosphorimaging as described in Subheading 3.3.1., steps 1–5.

3.3.3. Data Analysis: Gel Retardation 1. For direct titration it is possible to determine Kobs in an analogous manner to that described in Subheading 3.2.3. In this case, however, [ED]/[D]t = Countscomplex/Countsfree + Countscomplex = fraction of countscomplex where countscomplex and countsfree are the counts in the shifted (enzyme-DNA) and unshifted (free DNA) bands, respectively. The experimental data entered into the spreadsheet are fraction of countscomplex and the enzyme concentration [E]t for each titration point. 2. For competition binding the equations given in Subheading 3.2.5. are applicable. In this case, countscomplex [ED1] = [D1]t countsmax where countscomplex and countsmax are, respectively, the counts in the shifted band for each titration point at a particular competitor DNA concentration and for the control using large amounts of enzyme and no competitor DNA (see Subheading 3.2.4., step 5), where all the DNA is shifted into the complex. The known values for K1, [D1]t, [E]t, and countsmax are entered into SigmaPlot at the start of


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each calculation. The experimental data entered into spreadsheet columns are countscomplex and the competitor DNA concentration [D2]t for each titration point.

3.4. Fluorescence Anisotropy 3.4.1. KD Determination. Example: KD ≈40 nM 1. Set the fluorimeter to measure anisotropy in the “single-point polarization” mode. Each anisotropy value should be measured 10 times (measurement time, 5 s) and averaged (automatically carried out by the fluorimeter). Set the G-factor to “per measurement” (see Note 14). 2. An excitation wavelength of 530 nm is used with all the slits on the excitation monochromator set to 8. 3. Anisotropy is measured in the “L” format through the right photomultiplier with a 570-nm long-pass filter between the sample and the photomultiplier. Using a filter rather than a monochromator on the emission side greatly increases the light intensity at the photomultiplier and so allows measurement of lower concentrations of fluorophore. 4. Place a 0.5-mL fluorescence cuvet in the fluorimeter. The cuvet should not be removed from the fluorimeter or otherwise moved during the experiment. Add fluorescence binding buffer such that the final volume after step 3 will be 0.5 mL. A measurement temperature of 25°C is used. 5. Add duplex DNA, one strand of which is labeled with hexachlorofluorescein (Subheading 2.1., item 2) to give a final concentration of 10 nM (see Note 15). Note the anisotropy. 6. Add EcoRV endonuclease in small aliquots (see Note 16), using a microliter Hamilton syringe, to cover the range 0–400 nM. Mix thoroughly by gently withdrawing the contents with a plastic pipet tip and readding to the cuvet. After each addition, measure the anisotropy. 7. At the beginning (all oligonucleotide free in solution) and end (all oligonucleotide bound to endonuclease) of the titration, the fluorescence emission intensity should be noted (see Note 14).

3.4.2. Data Analysis 1. The fluorescence anisotropy measurements were carried out at [D] t ≈ KD (see Note 15). Under these circumstances, [E]f ≠ [E]t and so the simplified, hyperbolic, form of the binding equation cannot be used. Therefore, data must be fitted using the full quadratic binding equation (see Note 4). A solution, in terms of anisotropy, is –A 1 A A = Amin + max – min × ([D]t + [E]t + KD) – ([D]t + [E]t + KD)2 – (4 [D]t[E]t) [D]t 2

(

)[ (

√[

]

where A is the measured anisotropy; Amin is the anisotropy of free DNA, and is the Amax anisotropy of DNA when fully bound to the endonuclease. 2. The data should be fitted to the above equation using GraFit (11). This requires entry of A and [E]t into the spreadsheet provided by the software. [D]t is a con-

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stant (i.e., the concentration of oligonucleotide present at the start of the experiment). The titration consists of the addition of multiple aliquots of endonuclease to the cuvet, resulting in a dilution of the components. Correction should be made for the dilution of the endonuclease as aliquots are progressively added. Anisotropy, a ratio of fluorescence intensities, does not depend on concentration and is, therefore, unaffected by dilution. However, as [D]t is treated as a constant by this software it is not possible to compensate for its dilution. Therefore, the final dilution should not exceed about 10%. This may require endonuclease addition from several stocks of different concentration. GraFit also requires an estimate of Amin, Amax, and KD; suitable values for the first two can be obtained from the measured anisotropy values at the start (before endonuclease addition) and (after the final addition of endonuclease) at the end of the experiment. The software uses nonlinear regression analysis to calculate best-fit values of these three parameters. Note that the equation given above only holds if the fluorescence emission intensity of the free and protein-bound DNA are identical (see Note 14). This is determined in Subheading 3.4.1., step 6. 3. A representative titration curve is shown in Fig. 7.

4. Notes 1. With restriction endonucleases, it is often found that the binding of DNA and Mg2+ are rapid relative to subsequent events such as conformational changes, hydrolysis, and product release. This means that rate constants measured under single-turnover conditions are the same whether the reaction is initiated by adding Mg2+ to an endonuclease–DNA solution or adding the enzyme to a solution containing DNA and Mg2+. This should, however, be checked by trying both initiating methods. 2. The KD values for the EcoRV endonuclease and the oligonucleotides used in Subheadings 3.1.1. and 3.1.2. vary between 2 and 40 nM. The DNA concentration used in Subheadings 3.1.1. and 3.1.2. (25 × KD for a KD of 40 nM) and an endonuclease level 10 times higher ensures that ≥95.5% of the nucleic acid is bound to the protein. Many other concentrations of the two macromolecules formally meet this requirement. A useful combination, especially for proteins that are unstable, poorly soluble, and prone to form inactive tetramers and insoluble higher aggregates when free in solution is [D]t = 50 KD to100 KD and [E]t = 2 × [D]t. This results in at least 98% of the DNA being bound and avoids a large excess of the labile free protein. When bound to DNA, many endonucleases are both stabilized and protected from aggregation. For substrates with poor KD and kst (especially so-called “star” sites, where one of the bases in the recognition sequence is replaced by another natural base), dissociation rate constants are much greater than the cleavage rate constants. Therefore, multiple protein dissociations and associations occur prior to a productive cleavage event and so very poor substrates are never really cleaved under true single-turnover conditions. However, high concentrations of protein and nucleic acid ensure that the rate (ka × [DNA][E]) of association is as fast as possible and minimizes the


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Fig. 7. Representative binding isotherm determined by fluorescence anisotropy assay. An oligonucleotide composed of the complementary 14-mers 5'-HexTCCGGATATCACCT and 5'-AGGTGATATCCGGA was titrated with the EcoRV endonuclease. The binding buffer contained 50 mM Tris-HCl and 100 mM NaCl and the experiment was carried out at 25°C. The solid circles are experimental points. The KD value determined from the GraFit fit to the curve is 42 nM. chances that it contributes to the rate-determining step. Whatever levels of protein and DNA are selected, the rate constants should be independent of protein concentration if true single-turnover conditions apply. A useful empirical test is to measure rates at several protein concentrations to confirm this assumption. 3. For the equilibrium, kon Endonuclease + DNA Endonuclease–DNA KD = koff/kon koff the stability of a protein–DNA complex depends on koff. Most proteins have kon values of between 106 and 108 M–1s–1 enabling an estimate of the half-life (0.693/ koff) expected for an endonuclease–DNA complex at different KD values: KD = 1 µM (KA = 1 × 106/M–1); half-life between 0.01 and 1 s KD = 1 nM (KA = 1 × 109/M–1); half-life between 10 s and 10 m KD = 1 pM (KA = 1 × 1012/M–1); half-life between 100 m and 150 h 4. KD = [E]f [D]f/[ED] = ([E]t – [ED]) × ([D]t – [ED])/[ED] Therefore, the fractional saturation of the DNA with protein, [ED]/[D]t (the parameter measured using filter binding, gel shift, or fluorescence anisotropy), has a quadratic relationship to [E]t. However, under the condition [E]f = [E]t (which occurs if [E]t >> [ED]), KD = [E]t × ([D]t – [ED])/[ED] Here, [ED]/[D]t has a hyperbolic relationship to [E]t, simplifying data analysis. In order to achieve the condition [E]t >> [ED] and to obtain a satisfactory titra-

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tion it is also necessary to have [D]t < KD. DNA concentrations ≤15% KD are sufficient to satisfy the assumption [D]t < KD and give insignificantly different KD values whether the hyperbolic or quadratic binding equation is used. However, very low [D]t often gives binding curves of very poor quality. This may result from insufficient ligand stabilization of the protein by the nucleic acid. Therefore, a [D]t in the 5–15% KD range is recommended. Concentrations of protein are chosen so that the midpoint of the titration curve yields a protein concentration approx equal to the expected KD. Clearly, the KD will not be known prior to experimentation, emphasizing the need for an initial estimation, followed by a second, more accurate, determination. 5. Filter binding requires passage of the mixture through a nitrocellulose filter and a brief washing of the filter with buffer to remove excess unbound DNA. The entire process can be carried out in seconds. Gel retardation takes longer; about 1–2 min to load the samples and the same time to run them into the gel. Running the gel takes 30–60 min. Therefore, gel retardation can suffer, to a greater extent than filter binding, from dissociation of the protein–DNA complex. To some degree the problem is self-diagnostic, because it results in a smeared protein–DNA band rather than the tightly focused one which arises when no dissociation takes place. In these cases, KD determination may be achieved by measurement of the freeDNA band (16), but if this approach is used, an alternative method is best used to check the validity of the result. 6. As an example, consider an unmodified DNA with a KD of 0.1 nM and four modified DNAs with KD values that range from 1 nM to 1 mM. Direct equilibrium analyses are performed for the unmodified substrate and the modified site with KD = 1 nM. Equilibrium competition analyses are also performed for the four modified sites. If identical values of KD are obtained from both direct and competition analyses for the modified site with KD = 1 nM, the modified site serves as an internal control for the three modified sites which bind more weakly. 7. The relationship between fluorescence anisotropy, lifetime, and rotation is given by the Perrin equation (7,8): A = A0/(1 + τ/φ) where A is the measured anisotropy, A0 = intrinsic anisotropy (i.e., the anisotropy measured under conditions where no depolarization takes place), τ is the lifetime of the fluorescence excited state, and φ is the rotational correlation time, a measure of rotational diffusion that depends on the molar volume and, hence, the molecular mass attached to the fluorophore. The values of τ and φ must be balanced: a very short lifetime, relative to the correlation time, would lead to A = A0 ; a very long lifetime to A = 0. Use of this method requires 0 < A < A0. The τ value of approx 3 ns for hexachlorofluorescein attached to oligonucleotides is suitable for distinguishing between free and protein-bound oligonucleotides. The measured anisotropy is given by: A = (I|| – I⊥)/(⊥ I|| + 2I⊥)


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I|| and I⊥ are the intensities of the parallel and perpendicular components of the emitted light when parallel excitation is used. 8. The buffer components used for kst and KD determination will obviously vary somewhat depending on the enzyme under study and the particular experiment. However, for most DNA-binding proteins, including EcoRI, EcoRV, and BamHI restriction endonucleases there is pronounced nonlinearity in the dependence of log KD versus salt concentration (17) at salt concentrations ≤0.1 M, presumably because at these low salt concentrations, coupled protein–protein equilibria leading to aggregation (18) become significant. This phenomenon can produce misleading quantitative comparisons and thus salt concentrations below 0.1 M should be avoided. Bis-Tris propane is useful as it has two pK values (6.8 and 9), allowing pH-dependence studies with a single buffer. However, other buffers, of appropriate pK can equally well be used. Bovine serum albumin and DTT can help to stabilize certain restriction endonucleases, especially at low concentrations. With several DNA-binding proteins, including EcoRI endonuclease, aggregation and ultimately precipitation, in the absence of the stabilizing DNA substrate, is quite common at salt concentrations below 0.4 M. In these cases, intermediate dilutions should be made with buffers containing 0.6 M NaCl. Five percent glycerol, in the dilution buffers, also aids stability. 9. The “classic” gel-shift buffer, used to prepare and run nondenaturing polyacrylamide gel is 0.089 M Tris-borate, pH 8.0, containing 1 mM EDTA (19). Many protein–DNA complexes, particularly those of high affinity, are stable in this buffer. Therefore, it is often possible to incubate enzyme and DNA in a selected binding buffer and then carry out gel retardation in Tris-borate electrophoresis buffer without disturbing the equilibrium initially set up. However, the binding of restriction endonucleases to DNA depends on pH, salt concentration (NaCl or KCl), and divalent cations (Ca2+, a Mg2+ mimic that does not allow hydrolysis, often considerably strengthens binding) (18,20,21). Ideally it would be best to use identical incubation and electrophoresis buffers. We have obtained satisfactory gel retardation data using Tris-borate at pH 7.5. Replacing the EDTA with up to 5 mM CaCl2 is also not detrimental. However, many buffers are not suitable for electrophoresis, giving poorly resolved or smeared bands, especially if NaCl or KCl is added. 10. Gel retardation requires that the sample applied to the gel be denser than the electrophoresis buffer and so sinks into the well. The presence of 3% glycerol in the binding reactions ensures efficient sinking of the sample (18). If this method is used, it should be checked that glycerol does not perturb KD values (e.g., by carrying out filter binding ± glycerol. An alternative involves addition of half a volume (i.e., in this case, 5 µL to the 10 µL binding reaction) of 25% (w/v) sucrose solution following incubation and immediately prior to loading on the gel. This dilutes the sample, and in cases of weak binding, the associated fast koff rates may lead to some dissociation. However, if the binding is strong, the small dilution factor is unlikely to be a problem. It is best not to add the bromophenol blue marker to the solution of enzyme and DNA, as the dye can diminish (18) binding.

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11. Phosphorimaging is the best way to quantitate the amount of radioactivity in bands on gels. Phosphorimaging systems are now fairly standard in most laboratories. As an alternative autoradiography followed by scanning of the bands may be used, but it is more cumbersome and less accurate. 12. Fitting to the full kinetic model is actually the least robust for the unmodified enzymeoligonucleotide pair because the rate constants are so similar numerically that the program has difficulty partitioning among them. Thus, many closely spaced, and accurate, time-points should be taken and this will usually require rapid quench. 13. The total concentration of endonuclease and radiolabeled reference DNA (D1) are chosen to be at least fourfold over the predicted equilibrium dissociation constant (K1) for their interaction. [E]t and [D1]t in the range K1 to 30 K1 have been tested; the K2 (equilibrium dissociation constant for competitor DNA) is not affected significantly. 14. The change in anisotropy is only strictly related to the molecular mass of the fluorophore and hence the amount of oligonucleotide complexed with endonuclease, if the following two criteria are met. First, the detector response to the parallel and perpendicular polarized light must be identical. The G-factor measures this response, which is automatically corrected for, at each reading, with the fluorimeter used here. Second, the quantum yield of the free oligonucleotide and the enzyme-bound oligonucleotide must be the same (i.e., under identical conditions, the free and bound oligonucleotide should emit the same amount of light). If the quantum yield of the free and bound oligonucleotide varies the equation used to fit the data will not be valid (7,22,23). In the example shown in Fig. 7, the emission intensity of the free and bound oligonucleotide varies less than 10%. However, the use of other hexachlorofluorescein oligonucleotides and different buffers with the EcoRV endonuclease often results in large intensity changes (>10%). In these cases, fluorescence anisotropy cannot be used unless corrections are made (7,22,23). 15. Good results have been obtained using a DNA concentration about equal to the KD and varying protein concentrations from 0 to (20–30) KD. However, lower concentrations of DNA can also be used, although 1 nM represents the detection limit. DNA levels much higher than the KD should be avoided, as this promotes stoichiometric titration (where each protein molecule added binds to the nucleic acid), conditions under which accurate KD evaluation is not possible. 16. Purified endonucleases are often stored in buffers containing glycerol. Fluorescence anisotropy is very sensitive to viscosity and the addition of large amounts of glycerol during the titration adversely effects the measurement. Therefore, intermediate dilutions should be made with buffers lacking glycerol. This usually ensures that the amount of glycerol added to the fluorescence cuvet is negligible (≤2%). In the case of weak binding, when an enzyme may have to be added directly from the storage buffer, dialysis is recommended to remove the glycerol.

Acknowledgments The Newcastle group would like to thank G. Baldwin and S. Halford (Bristol) for the use of their quench-flow apparatus and help with the data


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shown in Fig. 4. B.A.C. is supported by the UK BBSRC and MRC. L.J.J. is supported by a grant (GM-29207) from the National Institutes of Health (USA). B.A.C. and L.J.J. have a Wellcome Trust biomedical research collaboration grant. References 1. Roberts, R. J. and Macelis, D. Rebase; the restriction enzyme database. http:// www. neb. com/rebase/rebase. html 2. Jen-Jacobson, L. (1995) Structural-perturbation approaches to thermodynamics of site-specific protein-DNA interactions. Methods Enzymol. 259, 305–344. 3. Jen-Jacobson, L. (1997) Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 44, 153–180. 4. Lesser, D. R., Kurpiewski, M. R., Waters, T., Connolly, B. A., and Jen-Jacobson, L. (1993) Facilitated distortion of the DNA site enhances EcoRI endonuclease DNA interactions. Proc. Natl. Acad. Sci. USA 90, 7546–7552. 5. Kurpiewski, M. R., Koziolkiewicz, M., Wilk, A., Stec, W. J., and Jen-Jacobson, L. (1996) Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of the EcoRI endonuclease with the phosphate at pGAATTC. Biochemistry 35, 8846–8854. 6. Lin, S. Y. and Riggs, A. D. (1972) lac Repressor binding to nonoperator DNA: detailed studies and a comparison of equilibrium and rate competition methods. J. Mol. Biol. 72, 671–690. 7. James, D. M and Sawyer, W. H. (1995) Fluorescence anisotropy applied to bimolecular interactions. Methods Enzymol. 246, 283–300. 8. Hill, J. J. and Roger, C. A. (1997) Fluorescence approaches to the study of protein-nucleic acid complexes. Methods Enzymol. 278, 390–416. 9. Powell, L. M, Connolly, B. A., and Drained, D. T. F. (1998) The DNA binding characteristics of trimeric EcoKI methyltransferase and its partially assembled dimeric form detected by fluorescence polarization and DNA footprinting. J. Mol. Biol. 283, 947–961. 10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 5.56–5.87 and 11.31–11.39. 11. GraFit (1998) Version 4.05, Erithacus Software, Staines, UK. 12. Scientist (1995) Version 2.0, MicroMath Software Inc., Salt Lake City, UT. 13. Sigma Plot (1996) Version 4.0 SPSS Inc, Chicago, IL. 14. Lesser, D. R., Kurpiewski, M. R., and Jen-Jacobson, L. (1990) The energetic basis of specificity in the EcoRI endonuclease–DNA interaction. Science 250, 776–786. 15. Jen-Jacobson, L., Lesser, D. R., and Kurpiewski, M. R. (1991) DNA sequence discrimination by the EcoRI endonuclease, in Nucleic Acids and Molecular Biology, Vol. 5 (Eckstein, F. and Lilley, D. M. J., eds.), Springer-Verlag, NY, pp. 141–170. 16. Carey, J. (1991) Gel retardation. Methods Enzymol. 208, 103–118. 17. Record, M. T., Jr., Anderson, C. F., and Lohman, T. M. (1978) Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins

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19. 20. 21. 22. 23.

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and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity. Quart. Rev. Biophys. 11, 103–178. Engler, L. E., Welch, K. K., and Jen-Jacobson, L. (1997) Specific binding by the EcoRV restriction endonuclease to its recognition site GATATC. J. Mol. Biol. 269, 82–101. Revzin, A. (1987) Gel electrophoresis assays for DNA–protein interactions BioTechniques 7, 346–355. Taylor, J. D. and Halford, S. E. (1989) Discrimination between DNA sequences by the EcoRV restriction endonuclease. Biochemistry 28, 6198–6207. Vipond, I. B. and Halford, S. E. (1995) Specific DNA recognition by EcoRV restriction endonuclease induced by Ca2+ ions. Biochemistry 34, 1113–1119. Eftink, M. R. (1997) Fluorescence methods for studying equilibrium macromolecule-ligand interactions. Methods Enzymol. 278, 221–257. Gutfreund, H. (1995) The role of light in kinetic investigations, in Kinetics for the Life Sciences Receptors, Transmitters and Catalysts. Cambridge University Press, Cambridge, pp. 291–295.

Intrinsic Fluorescence

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33 Analysis of DNA–Protein Interactions by Intrinsic Fluorescence Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale 1. Introduction Changes in the fluorescence emission spectrum of a protein upon binding to DNA can often be used to determine the stoichiometry of binding and equilibrium binding constants; in some cases the data can also give an indication of the location of particular residues within the protein. The experiments are generally quick and easy to perform, requiring only small quantities of material (1). Spectroscopic techniques allow one to measure binding at equilibrium (unlike, for example, gel retardation assays and other separation techniques that are strictly nonequilibrium methods). Fluorescence is one of the most sensitive of spectroscopic techniques, allowing the low concentrations (typically in the nanomolar to micromolar range) required for estimation of binding constants for many protein–DNA interactions. Considerable care, however, needs to be exercised in the experiment itself and in the interpretation of results. The fundamental principles of fluorescence are discussed briefly in the remainder of the Introduction. A molecule that has been electronically excited with ultraviolet (UV)/visible light can lose some of the excess energy gained and return to its ground state by a number of processes. In two of these, fluorescence and phosphorescence, this is achieved by emission of light. Phosphorescence is rarely observed from molecules at room temperature and will not be considered further. Although electrons can be excited to a number of higher-energy states, fluorescence emission in most cases only occurs from the first vibrational level of the first excited state. This has two implications for the measurement of fluorescence emission spectra. First, some of the energy initially absorbed is lost prior to emission, which means that the light emitted will be of longer wavelength From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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(i.e., lower energy) than that absorbed. This is known as the Stoke’s shift. Second, the emission spectrum and therefore the wavelength of maximum fluorescence will be independent of the precise wavelength used to excite the molecule. Thus, for tyrosine, the wavelength of the fluorescence maximum is observed around 305 nm, regardless of whether excitation is at the absorption maximum (approx 278 nm) or elsewhere in the absorption band. Of course, the fluorescence intensity will change as a consequence of the difference in the amount of light absorbed at these two wavelengths. The fraction of light emitted as fluorescence compared to that initially absorbed is termed the quantum yield. The value of the quantum yield for a particular fluorophore will depend on a number of environmental factors such as temperature, solvent, and the presence of other molecules that may enhance or diminish the probability of other processes deactivating the excited state. The deactivation or quenching of fluorescence by another molecule, either through collisional encounters or the formation of excited-state complexes, forms the basis of many of the fluorescence studies on protein–DNA interactions. The study of protein–nucleic acid interactions is greatly simplified by the fact that all detectable fluorescence arises from the protein, all four of the naturally occurring DNA bases being nonfluorescent by comparison. Tyrosine and tryptophan residues account for almost all the fluorescence found in proteins. As a general rule, when both residues are present, the emission spectrum will be dominated by tryptophan, unless the ratio of tyrosines to tryptophans is very high. The quantum yield of a tyrosine residue in a protein compared to that observed in free solution is generally very low, illustrating the susceptibility of tyrosine to quenching. Tryptophan residues are highly sensitive to the polarity of the surrounding solvent, which affects the energy levels of the first excited state with the result that the emission maximum for tryptophan can range from 330 nm in a hydrophobic environment to 355 nm in water. Thus, in proteins containing only one tryptophan, the general environment surrounding the residue can be ascertained. Tryptophan fluorescence, like that of tyrosine, can be quenched by a number of molecules, including DNA. Unlike tyrosine, the emission maximum can also change if tryptophan is involved in the interaction and this can also be used to monitor DNA binding (2). The extent to which the fluorescence of a protein is quenched by DNA is proportional to the concentration of quencher. As quenching is the result of the formation of a complex between the protein and the DNA, the extent of quenching is proportional to the amount of bound protein. Thus by determining the extent to which the protein fluorescence is quenched when fully bound to DNA (i.e., at saturation), the fraction of bound and free protein at any point in a titration can be determined. From these data, the stoichiometry and binding constant of the interaction can often be obtained. Note that to establish an


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accurate stoichiometry, a high concentration of protein is preferred when titrating with DNA (i.e., well above the Kd of the complex) to ensure stoichiometric binding. To establish the binding constant itself, one should be working at much lower concentrations of protein so that at the stoichiometric point, there is a measurable concentration of unbound protein. In the case of protein– DNA interactions having a low Kd, this may not be possible. If fluorescence quenching is being used to follow DNA binding, it is vital to take account of sample dilution, as well as the increased absorption of the sample as DNA is titrated in. The latter effect is known as the inner filter effect and arises from the absorption of the excitation beam (and generally to a lesser extent, the emission beam) on passing through the sample (see Fig. 1). One should aim to keep the absorption of the sample (at the excitation wavelength) as low as possible, although absorbances up to 0.2 can normally be corrected without too much difficulty. A small-pathlength cell will also help (if rectangular, the excitation beam should pass through the smallest path). Ideally, the absorption of the sample at the excitation and emission wavelengths (Aex and Aem ) should be measured for each point in the titration (if not, one can calculate these values from the known concentrations of protein and nucleic acid at each point). For normal right-angled geometry of observation, the corrected fluorescence Fcorr can be obtained from the observed fluorescence Fobs by the formula Fcorr = Fobs × 10(Aex/2 + Aem/2)

(1)

Often, the value of Aem is small enough to ignore (for a detailed treatment of the inner filter correction, see ref. 3). Note that it is equally important to correct for the inner filter effect whether titrating protein into DNA or vice versa. The following method deals only with the determination of DNA binding curves by intrinsic fluorescence quenching. However, fluorescence anisotropy can also be used if the molecular size of the complex is sufficiently different from the free protein; for example, to investigate proteins that bind cooperatively to DNA (4). Time-resolved fluorescence techniques are also advantageous (for measurements of fluorescent lifetimes or rotational correlation times) but require sophisticated instrumentation (5). The use of intrinsic fluorescence, as a method for investigating protein– DNA interactions, is widespread. For example, both binding parameters (Kobs) and stoichiometric ratios have been derived for the interaction of the HIV-1 nucleocapsid protein NCp7 with the natural primer tRNA3Lys and other related RNA molecules (6). Similarly, estimates of binding constants have been determined for the interaction of human replication protein A (hRPA) with singlestranded homopolynucleotides (e.g., poly[dT] and poly[dA]) (7). Furthermore, both steady-state and time-resolved fluorescence have been used in a binding study of the single-stranded DNA-binding protein of phage φ29 (8).

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Fig.1. Schematic representation of the inner filter effect in fluorescence, showing the effect of high concentration on the absorbance of the excitation beam.

2. Materials 1. Reagents used in buffer solutions should be of the highest purity available and the solutions prepared in doubly-distilled water. The buffer should have negligible absorbance in the 260- to 300-nm wavelength range and should not be used if it shows any fluorescence in the region 290–400 nm. High-molecular-weight


2. 3. 4.

5.

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ions should be avoided. Phosphate buffer should not be used if tyrosine fluorescence is being monitored. Stock solutions of protein and DNA should be divided into small aliquots and stored at –20°C, assuming this is not detrimental to the protein. High-quality quartz cuvets with all four faces polished (see Notes 1 and 2). Most commercially available fluorimeters allow scanning by both the excitation and emission monochromators and are suitable for use in these studies. Cell compartments that can be thermostatically controlled are preferable (see Notes 3 and 4). We routinely use a Perkin-Elmer LS 50B fluorimeter. An on-line computer is normally linked to the fluorimeter, which allows fluorescence spectra to be recorded and analyzed. We routinely use the software provided with the Perkin-Elmer LS 50B fluorimeter “FL WinLab.”

3. Method The method described in this section assumes that nothing is known concerning the fluorescence properties of the protein or its complex with DNA. Consequently, the initial steps described in Subheading 3.1. are concerned with characterizing some of the fluorescence properties of the two species such that the optimal conditions for obtaining accurate and reliable data can be obtained. Subheading 3.2. describes the procedure for obtaining data for a protein that is quenched by DNA, which results only in a decrease in fluorescence intensity, and how these data can be used to obtain information on binding. Several variations of the method are mentioned in the Subheading 4.

3.1. Preliminary Experiments 1. Switch on the fluorimeter and allow 10 min for the components to stabilize. Set the excitation and emission slits to intermediate values (e.g., 10 nm bandpass). 2. Fill the cuvet with protein solution (see Note 5). Allow time for the solution to equilibrate to the temperature of the compartment. To prevent local heating of the solution or possible photodecomposition, the excitation shutter should be kept closed, except when taking measurements. 3. If the absorption spectrum of the protein is known, set the excitation wavelength to that corresponding to the absorption maximum between 265 and 285 nm; if no peak exists, the protein does not contain tyrosine or tryptophan residues and will not fluoresce. If the absorption spectrum is unknown, set the excitation wavelength to 280 nm. 4. Open the excitation shutter and quickly scan the emission between 285 and 400 nm, looking for the wavelength at which a maximum value for the intensity is given on the readout. Return the emission monochromator to this wavelength. 5. Find the excitation wavelength maximum between 265 and 285 nm in the same manner, with the emission monochromator set at the wavelength of maximum fluorescence. Note: The aforementioned “FL WinLab” software allows simultaneous scanning of both the excitation and emission wavelengths, in what is

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6.

7.

8.

9.

10.

Carpenter, Oliver, and Kneale termed a “3D” scan. This allows both the excitation and emission maxima to be determined in one experiment. However, for proteins, this is not normally necessary. With both the excitation and emission wavelengths set at their peak values, adjust the instrument to give a reading corresponding to about 90% of the full scale. Narrow slit widths and a lower amplification (expansion factor, gain) are preferred and a compromise between the two may have to be found (see Note 6). Determine the emission spectrum by scanning the emission monochromator over the entire wavelength range over which fluorescence occurs. A scan speed of 60 nm/min is generally suitable. Add a small aliquot of a concentrated DNA solution to the cuvet such that the concentration of DNA is in excess. Mix and immediately check the fluorescence emission at the emission maximum of the protein. Check several times over the next few min until a consistent reading is obtained. Allow this time for equilibration in subsequent experiments. Do not adjust slit widths or the amplication. Obtain an emission spectrum and compare with that obtained for the protein only. If fluorescence quenching is suspected, make sure that allowance for sample dilution has been made. If an inner filter correction is required, measure the absorbance of the sample in a spectrophotometer (in the same cuvet) and correct the observed fluorescence as discussed in Subheading 1. Add aliquots of DNA until there is no further change in fluorescence intensity in the emission spectrum (see Notes 7 and 8).

3.2. Protein-DNA Titrations 1. Examine the emission spectrum of the free protein. If it is characteristic of tyrosine fluorescence, check for interference from the Raman band (see Note 9). If it is characteristic of tryptophanlike, check for tyrosine contributions that may be masked (see Note 10). 2. Examine the emission spectrum of the protein bound to DNA. The titration method described in the following passage is particularly applicable when the only change in the spectrum is a change in fluorescence intensity. Several variations of this method are described briefly in Notes 11 and 12 including an example where the emission spectrum of the protein shifts on binding DNA. 3. Accurately determine the concentration of protein and DNA solutions by UV spectroscopy. Because we are titrating DNA into protein, try to use a stock concentration of DNA, which is at least 20 times the concentration of protein used in the experiment multiplied by the estimated stoichiometric ratio; for example, if the protein concentration used is 10 µM and the estimated stoichiometry is 5 bases per protein, then the DNA concentration should be at least 20 × 10 µM × 5 = 1000 µM (1 µM). This would mean that the dilution of the original protein solution will be only 5% at the stoichiometric point. 4. Using the protein solution set up the instrument as described in steps 1–6 of Subheading 3.1. If measuring tyrosine fluorescence, use an excitation wavelength near the maximum. This wavelength can also be used for tryptophan excitation if tyrosine fluorescence is insignificant, otherwise use an excitation wavelength of 295 nm.


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5. Run a buffer blank and check that the profile of the emission spectrum is consistent with that previously obtained. Subtract this spectrum from subsequent spectra if this can be done automatically. 6. Set the emission monochromator to the emission wavelength maximum and ensure that the readout is about 90% of its maximum value. Note the value. 7. To begin the titration, add a small aliquot from the stock DNA solution to the protein in the cuvet. Mix and allow the sample to equilibrate (use the time period determined earlier) before taking a reading. The aliquots should be sufficiently small such that the protein is still greatly in excess and a linear change is observed as more DNA is added. 8. Continue to add the same quantity of DNA for 8–10 points. If changes are still approximately linear at this stage, gradually increase the volume of the DNA added, noting the total amount added at each point. 9. When the change in intensity begins to deviate significantly from linearity, decrease the size of the aliquot so that more data points are obtained in this region. 10. As quenching approaches the maximum, larger aliquots of DNA can be added. Continue until no change in quenching is observed for several points. 11. After the last point, check that the emission spectrum of the complex is consistent with that previously obtained for the bound protein. 12. Remove the sample, wash the cuvet thoroughly, and run a blank spectrum consisting of cell plus buffer. This should have negligible or no fluorescence. Subtract any value at the emission wavelength maximum from the data points, if not already done automatically (see Note 9). 13. For each data point, calculate the fluorescence quenching (Q= 100 [F0–F]/F0 , where F is the measured fluorescence and F0 is the fluorescence in the absence of DNA), having made any corrections for inner filter effects and dilution of the sample. Also calculate the nucleotide concentration at each point (N = ny/[n + x], where n is the total volume of DNA added up until that point, x is the initial volume of sample in the cuvet and y is the molar concentration of DNA stock solution). From the DNA concentration, calculate R, which is the ratio of the concentration of DNA to that of protein. (For polynucleotides, it is usual to express N as the concentration of nucleotides; for short synthetic duplexes, it is more usual to use the molar concentration of the duplex). 14. Plot a graph of Q against R (or N). If only one mode of binding is occurring, the graph should look like one of the curves shown in Fig. 2. If the “break point” in the titration is sharp, it indicates a high value for the binding constant (i.e., a small dissociation constant compared with the protein concentration used). Conversely, too weak a binding constant (or too dilute a protein solution) will give rise to a smoothly rising curve with no apparent break point. 15. The stoichiometry of binding is the value of R at which the slope obtained from the initial linear range of the titration crosses the horizontal line defined by Qmax, at which no further change in intensity occurs. 16. Further information can be extracted from the binding curve by fitting it to an appropriate model. In the simplest case of a bimolecular interaction (P + N =

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Fig. 2. A graph of fluorescence quenching against DNA:protein ratio (R). The curves illustrate the addition of DNA (in this case, a polynucleotide) to protein (10 µM). The theoretical binding curve for infinitely strong binding (upper curve) shows a stoichiometry of five nucleotides bound per protein. Typical curves are shown for binding constants of 107 (䊏), 106 (䉬), and 10 5 (䉱) per molar. PN), then a useful expression to estimate the binding constant is K = [P0]θ/(1–θ)2 where θ is the fraction of bound protein at the stoichiometric point and [P0 ] is the total protein concentration in the cuvet. This expression also applies to more complex cooperative binding along a linear DNA lattice (9), assuming the cooperativity is sufficiently high, when K becomes equal to the apparent binding constant (and approx the product of the cooperativity factor and the intrinsic binding constant for one site). For a more extensive discussion of complex DNA binding equilibria, see ref. 10.


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4. Notes 1. Most fluorimeter cell compartments are designed to take cuvets with a 1-cm pathlength (distance between opposite faces) and usually require 2.5–3.0 cm 3 of sample for measurements. If smaller sample volumes are required, then reduced volume cuvets similar to those used in absorption studies but suitable for fluorescence work can usually be obtained from most cuvet suppliers. Also, most suppliers are prepared to construct cuvets to your own specifications (at a cost). The major requirement is that the sample be located in the center of the cell (assuming standard right-angle observation). Cuvets holding as little as 300 µL have been successfully used by the authors. 2. Care should be taken when handling fluorescence cuvets, as both fingerprints and scratches can introduce significant artifacts into the experiment. After use, cuvets should be thoroughly washed with distilled water and a mild detergent, if necessary. If greatly contaminated, then immerse the cells in a 50:50 mix of ethanol with sulfuric acid (4 M) for several hours and then rinse thoroughly with water. 3. If the fluorimeter is not equipped with a magnetic stirrer unit, adequate mixing can usually be achieved by gently drawing the solution in and out through a plastic pipet tip. Avoid introducing bubbles into the sample, as this can both denature the protein and cause light scattering. For accurate measurements, temperature control is essential, as fluorescence is highly sensitive to temperature. 4. If the use of a reduced-volume cell prevents the use of commercially available magnetic fleas, then substitutes can be made as follows: (1) Seal the narrow end of a Pasteur pipet or micropipet by heating it in the flame of a Bunsen burner. (2) Insert a small length of iron wire (cut up a paperclip) and shake it down to the sealed end. (3) Cut the pipet just above the wire using a glass cutter and seal the open end in the flame. These fleas should only be used once, as some rusting occurs with time. 5. Fluorescence intensity is only proportional to concentration when the absorbance is no greater than 0.1 absorbance units at the excitation wavelength selected. If a molar extinction coefficient for the protein is known, use this to calculate a suitable protein concentration. Remember that the absorption bands for proteins and nucleic acids overlap, and in titrations, the contribution of the nucleic acid to the overall absorption must be considered. 6. Although it is preferable to have narrow excitation and emission slit widths and a low amplification factor, there may be a need to compromise in order to obtain a stable reading. For proteins displaying tyrosine fluorescence, the small wavelength difference between the excitation and emission maxima suggests that it would be better to maintain narrow slits and increase the signal amplification. For proteins dominated by tryptophan fluorescence, the greater the difference between the excitation and emission wavelengths, the greater the feasibility of increasing the slit widths and maintaining a lower amplification. In general, when measuring emission spectra, it is better to use a narrow emission slit width and widen the excitation slit width. For broad-banded spectra such as that seen with tryptophan, both slits can be widened.

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7. If no changes in the emission spectrum of the protein are observed when DNA is added (after inner filter and dilution corrections if necessary), then either the protein is not binding to DNA or binding cannot be detected by this procedure and will need to be assessed by another method, such as fluorescence anisotropy or the use of an extrinsic probe (see Chapter 18). 8. Note the molar ratio of DNA: protein at which no further changes occur. This will provide a rough guide for future experiments. 9. Tyrosine emission can often be confused with Raman scattering of light, which occurs around 305 nm when an excitation wavelength of 280 nm is used. The presence of the Raman band can be assessed by measuring the emission spectrum using a different excitation wavelength. The fluorescence emission spectrum is independent of excitation wavelength, whereas Raman scattering occurs at a constant wave number (= 1/λ, in cm–1) from that used for excitation and will shift in the same direction as the change in excitation wavelength. The contribution of the Raman band to the overall intensity of the signal can be assessed by running an emission spectrum of a buffer blank. Automatically subtract out this spectrum from subsequent spectra where possible. Note: The “FL WinLab” software offers a prescan mode, in which the Raman peak can be identified automatically. Other fluorescence software packages may offer a similar facility. 10. To check the contribution tyrosine may make to a fluorescence emission spectrum dominated by tryptophan, run an emission spectrum using an excitation wavelength of 295 nm. At this wavelength, only tryptophan emission will be observed. If the emission spectrum is unchanged, then it can be concluded that the contribution from tyrosine residues is negligible. (Of course, the intensity will be lower, as tryptophan absorption is greater at 280 nm than it is at 295 nm.) 11. In cases where both the emission maximum shifts and the fluorescence intensity is quenched, the method described can be used provided that an emission wavelength is chosen outside the wavelength region overlapped by the emission spectra of the free and bound protein. Alternatively, the ratio of the intensity of the emission maxima of the free and bound proteins can be followed (for an example, see ref. 2). The use of a ratio method means that the dilution factor and inner filter correction can usually be ignored, although, strictly speaking, the ratio is not a linear function of degree of binding. 12. In some cases it may be preferable to titrate DNA with protein (for an example method, see ref. 11). The procedure is similar to that given here, but in this case the experiment should be repeated by adding protein to the buffer in the absence of DNA as a reference. Subtraction of the two curves should yield a clear binding curve (see Fig. 3). (For a discussion of the merits of whether to titrate protein with DNA or vice versa, see ref. 4). We have found that in some cases, different results can be found dependent on the direction of the titration; this can occur when the fluorescence changes observed include contributions from protein–protein interactions accompanying DNA binding in addition to (or instead of) contributions from the interaction with the DNA itself.


501 501

Fig. 3. (A) Titration of an oligonucleotide with fd gene 5 protein. Fluorescence (305 nm) of increasing concentrations of protein (P) is measured in the presence (lower curve) and absence (upper curve) of DNA. The difference between these two curves is plotted in (B) along with the theoretical binding curve. In this experiment, the starting concentration of DNA was 16.7 µM.

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References 1. Harris, D. A. and Bashford, C. L. (eds.) (1987) Spectrophotometry and Spectrofluorimetry: A Practical Approach. IRL Press, Oxford, UK. (Chapters 1 and 4 are particularly relevant.) 2. Kneale G. G. and Wijnaendts van Resandt, R. W. (1985) Time resolved fluorescence of the Pf1 bacteriophage DNA-binding protein: determination of oligo- and polynucleotide binding parameters. Eur. J. Biochem. 149, 85–93. 3. Birdsall, B., King, R. W., Wheeler, M. R., Lewis, C. A., Goode, S. R., Dunlap, R. B. et al. (1983) Anal. Biochem. 132, 353–361. 4. Carpenter, M. L. and Kneale, G. G. (1991) Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT). J. Mol. Biol. 217, 681–689. 5. Greulich, K. O., Wijnaendts van Resandt, R. W., and Kneale G. G (1985) Time resolved fluorescence of bacteriophage Pf1 DNA-binding protein and its complex with DNA. Eur. Biophys. J. 11, 195–201. 6. Mély, Y., de Rocquigny, H., Sorinas-Jimeno, M., Keith, G., Roques, B. P., Marquet, R., et al. (1995) Binding of the HIV-1 nucleocapsid protein to the primer tRNA3Lys in vitro, is essentially not specific. J. Biol. Chem. 270, 1650–1656. 7. Kim, C. and Wold, M. S. (1995) Recombinant human replication protein A binds to polynucleotides with low cooperativity. Biochemistry 34, 2058–2064. 8. Soengas, M. S., Mateo, C. R., Salas, M., Acuña, A. U., and Gutiérrez, C. (1997) Structural features of φ29 single-stranded DNA-binding protein. J. Biol. Chem. 272, 295–302. 9. Kelly, R. C., Jensen, D. E., and von Hippel, P. H. (1976) Fluorescence measurements of binding parameters for bacteriophage T4 gene 32 protein to mono-, oligo, and polynucleotides. J. Biol. Chem. 251, 7240–7250. 10. McGhee, J. D. and von Hippel, P. H. (1974) Theoretical aspects of DNA–protein interactions: cooperative and non-cooperative binding of large ligands to a onedimensional homogeneous lattice. J. Mol. Biol. 86, 469–489. 11. Alma, N. C. M., Harmsen, B. J. M., de Jong, E. A. M., Ven, J. V. D., and Hilbers, C. W. (1983) Fluorescence studies of the complex formation between the gene 5 protein of bacteriophage M13 and polynucleotides. J. Mol. Biol. 163, 47–62.

Circular Dichroism

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34 Circular Dichroism for the Analysis of Protein–DNA Interactions Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale 1. Introduction The asymmetric carbon atoms present in the sugars of nucleotides and in all the amino acids (with the exception of glycine) results in nucleic acids and proteins displaying optical activity. Further contributions to the optical activity of the polymers result from their ability to form well-defined secondary structures, in particular helices, which themselves possess asymmetry. As a consequence, circular dichroism (CD) has found widespread use in secondary structure prediction of proteins (1). Similar studies, though less widespread, have sought to correlate structural parameters of DNA with their CD spectrum (2), with some success particularly in assigning quaternary structures to nucleic acids (e.g., in the case of DNA triplexes and G-quartet mediated structures) (3,4). It follows that the disruption of secondary structure by, for example, denaturation or ligand binding can be usefully followed by circular dichroism. Plane polarized light can be resolved into left- and right-handed circularly polarized components. Circular dichroism measures the difference in the absorption of these two components, ∆ε = εL – εR

(1)

where ε is the molar extinction coefficient the left (L) and right (R) components (see Note 1). When passing through an optically active sample, the plane of polarized light is also rotated, which means that the emerging beam is elliptically polarized. Thus, CD is often expressed in terms of ellipticity (θλ, in degrees) or molar ellipticity ([θ]λ, in degrees cm2/dmol). (M–1/cm–1) for

[θ]λ = 100 θλ/cl

(2)

From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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where c is the molar concentration and l is the pathlength (in cm). The two expressions are interconvertible via the expression [θ]λ = 3300 ∆ε. The overlap of the ultraviolet (UV) absorption bands of nucleic acids and proteins means that CD studies of protein–DNA interactions can be complicated by the contributions observed from both components. This is particularly true for wavelengths less than 250 nm. In practice, CD spectra between 250 and 300 nm are dominated by that of the nucleic acid, the contribution arising from the aromatic chromophores of the protein being weak by comparison. Changes in conformation can usually be attributed to the polynucleotide, as the random distribution of aromatic amino acids means a large conformational change throughout the protein would be required to cause a significant change in the CD spectrum. The low-molar ellipticity of polynucleotides means that for accurate CD measurements, high concentrations (10–4 M to 10 M) of nucleotide are required. For this reason, circular dichroism is not generally used to determine binding constants of protein–DNA interactions. However, circular dichroism can be used to obtain accurate values for the stoichiometry of protein–nucleic acid interactions (5), and in the case of the bacteriophage, the fd gene 5 protein was used to show the existence of two distinct binding modes (6). Circular dichroism has also been used to show that conformational changes induced by the bound Lac repressor are different for operator DNA and for random sequence DNA (7). Similar studies on the Gal repressor demonstrated the involvement of the central G-C base pairs of the operator sequence in repressor-induced conformational changes (8). Studies on the interaction of the cro protein of bacteriophage λ have also revealed different conformational changes for specific and nonspecific DNA binding (9). Despite the apparent lack of any direct interaction of the central base pair of the operator sequence with the cro protein, base substitution at this site was shown to affect the CD spectrum considerably. Some additional examples in which CD has been used to examine protein– DNA interactions include the SRY-related protein Sox-5 (10), the type IC DNA methyltransferase M. EcoR124I (11), and the bacteriophage Pf3 singlestranded DNA-binding protein (ssDBP) (12). It should be emphasized that circular dichroism provides complementary data to other spectroscopic techniques such as fluorescence, because each technique can monitor different components of the interaction. For this reason, even the apparent stoichiometry of binding estimated by each technique could be significantly different despite it being measured under the same solution conditions (5). 2. Materials 1. A high-quality quartz cell with low strain is required for accurate measurements. The cell pathlength will depend on the absorption properties and concentration of

Circular Dichroism

2.

3.

4. 5.

6.

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the sample. Cells with pathlengths between 0.05 cm and 1 cm are often used, depending on the CD signal to be measured and the absorption of the sample (including the buffer). A pathlength of 1 cm is usually recommended for measurement of the DNA signal in the vicinity of 275 nm, where the signal is weak and buffer absorption is negligible. Buffers should be prepared using high-quality reagents and water. Use buffers that have low absorbance in the wavelength region of interest. Tris-HCl, perchlorate, and phosphate are routinely used. Stock solutions of appropriate protein and nucleic acid solutions in the same buffer. The protein should be as concentrated as possible to minimize dilution during the titration. If a synthetic DNA fragment containing the recognition sequence is to be used, it should be close to the minimum size required for binding to maximize the change in CD signal. To avoid denaturation and degradation, keep concentrated solutions of protein and DNA frozen in small aliquots, assuming it has been established that this procedure does not damage the protein. A supply of dry nitrogen (oxygen free). (+)10-Camphor–sulfonic acid at a concentration of 0.5 mg/mL, used as a calibration standard. Check the accurate concentration by UV spectroscopy using a molar extinction coefficient of 34.5 at 285 nm (1). A circular dichroism spectrometer (spectropolarimeter). We currently make use of a Jasco J720 spectrometer.

3. Methods For most proteins, there is only a weak signal from aromatic amino acids in the region of the CD spectrum between 250 and 300 nm compared to that seen for nucleic acids. Experiments involving the addition of protein can thus be conveniently carried out in this wavelength range, as described in the following. Below 250 nm, both proteins and DNA have optical activity and any experiments here may require resolution of the spectrum into protein and DNA components (see ref. 13 for an example of where a mutant protein has been used to assign and interpret overlapping CD bands). 1. To prevent damage to the optics, flush the instrument with nitrogen for 10–15 min before switching on the lamp. Continue to purge the instrument for the duration of the experiment (see Note 2). 2. Switch on the lamp and allow the instrument to stabilize for 30 min before making any measurements. 3. While waiting for the instrument to warm up, measure the UV spectrum of both the DNA and protein and calculate the concentration of the stock solutions from their extinction coefficients. The stock solution of protein should be as high a concentration as possible, to minimize corrections for dilution in subsequent titrations. 4. Measure the UV absorbance of the cell to be used in the CD experiment against an air blank (see Note 3).

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5. Using the data from steps 3 and 4, determine the concentrations of DNA and protein that can be used in the experiment such that the total absorbance of all components including the cell is 1000KD) and with the injection of low amounts of protein). The total number of moles of added protein is then much less than the number of moles of binding sites available and virtually all protein molecules are bound to DNA, such that ∆[P]i,bound approximately equals ∆[P]i,total. Each injection of protein, ∆[P]i,total thus produces an approximately equal change in heat, ∆qi,app. If the overall fractional saturation is still low after completion of a series of injections, then the average may be taken and Eq. 3 becomes 1 m 1 m ∑∆qi = ∑ ∆qi + ∆qi,ns m i=1 ,app m i=1

(

=

)

1 m ∑ ∆[P]i,bound + Vcell ∆Happ m i=1

(

(6)

)

where m is the number of injections. If low amounts of protein are injected, aggregation effects and nonspecific binding are also minimized.

1.3. The DSC Experiment In a DSC experiment, the heat capacity of a macromolecule is measured as a function of temperature. Typically, two thermally insulated cells, a sample cell containing the macromolecule of interest in buffer and a reference cell containing buffer, are electrically heated at a known rate. At a temperature-induced transition, which is typically endothermic, the temperature of the sample cell will lag behind that of the reference cell. An electrical feedback mechanism is used to maintain the reference cell at the same temperature as the sample cell. This amount of compensatory electrical power (in units of J/s or Watts) at temperature T divided by the heating rate is the apparent difference in heat capacity between the cell containing the sample and the reference cell, ∆Cp,app(T) (in units of J/K). Because the sample containing cell has a smaller volume fraction of buffer as compared to that in the reference cell, the partial molar heat capacity of the dissolved macromolecule Cp,f(T) at temperature T (J/mol/K) is given by

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Read and Jelesarov CTp,φ(T) =

Cp,buffer (T)Vφ˚ M∆Cp,app (T) Vbuffer

m

(7)

where Cp,buffer(T) and Vbuffer are the partial molar heat capacity and molar volume of buffer, respectively. Vf˚ and M are the partial molar volume and the molar mass of the macromolecule, respectively, and m is the mass of macromolecule in the sample cell. The excess molar heat capacity function is the heat absorbed in the melting transition above that of the intrinsic heat capacity of the macromolecule. Integration of the excess molar heat capacity function with respect to temperature yields the enthalpy of the melting transition: T2

∆H = 兰具Cp(T)典dT

(8)

T1

The intrinsic heat capacity of the initial (folded) and final (unfolded) states of the macromolecule must be estimated within the melting transition. For monomeric proteins, the heat capacity of the folded state is an approximate linear function of temperature (13,14) and the unfolded state is a shallow parabolic function of temperature (13). The relationship of the partial molar heat capacity to the excess molar heat capacity functions are schematically shown in Fig. 1.

1.4. Preliminary Characterization of the Sox-5–DNA Interaction It must be emphasized that the in vitro interaction of the protein with DNA must be characterized prior to the calorimetry experiments. Preliminary knowledge of the number of (independent) binding sites, the stoichiometry of the interaction, and the magnitude of the association constant are all required for the optimal design of the ITC experiments (see Subheading 1.2.). For DSC, the experiments must be performed with a fully associated complex (at low temperature) and therefore at a concentration well above the estimated KD. Furthermore, the protein–DNA complex and its components need to be soluble at this concentration, even at raised temperatures, and all temperature-induced conformational transitions must be fully reversible. Knowledge of the mode of interaction is also required to obtain van Hoff enthalpies from the DSC data. Site-selection and DNAse I footprinting assays (Chapter 3) defined the Sox-5 target sequence as 5’-AACAAT-3' within a DNAse I protected region of approx 14 nucleotides on both DNA strands (15,16). This determined the sequence and size of the DNA duplexes that were to be used. A circular permutation assay showed that the binding of Sox-5 to these duplexes was able to introduce a DNA bend of similar magnitude to that obtained with the complete

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Fig. 1. Schematic representation of (A) the partial molar heat capacity function as would be observed for the melting of a single domain monomeric protein. Over the transition region the intrinsic heat capacity function is an interpolation of the folded and unfolded states, weighted in proportion to their relative contributions (dashed line). (B) replots the Cp/T function above that of the intrinsic heat capacity of the system. This is known as the molar excess heat capacity function.

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Sox-5 protein (6,16). The dissociation constant and the stoichiometry of the Sox-5–DNA interaction were determined from circular dichroism (CD) and gel-shift assays (Chapters 2 and 34): 1. The binding of Sox-5 to a 12-bp DNA duplex (10 µM) was followed by CD, monitoring the ellipticity of the positive DNA peak at 280 nm upon successive additions of the protein. The CD data were fitted to a 1:1 binding model with an estimated KD of lower than 100 nM (6). This same estimate was obtained at temperatures between 10°C and 37°C, suggesting no significant variation of binding constant with temperature. 2. A gel-shift assay using a radioactively labeled 12-bp DNA duplex (at 1 nM) was titrated in molar excess with Sox-5 (6). This indicated that the primary binding site is characterized by a KD of about 35 nM at 4°C. No secondary protein binding was observed with 10 µM (i.e., at least two orders greater than the primary binding site). Thus, for the DSC measurements, typically at a concentration of 100– 500 µM complex, the preformed Sox-5–DNA complex is fully associated and the effect of secondary binding is negligible. Given the estimated KD value, the ITC measurement of a full binding isotherm under optimal conditions requires a DNA concentration of between approx 0.35 and 3.5 µM (see Subheading 1.2.). Preliminary titration experiments however showed only a small heat effect of association (a result of the temperature dependency of ∆Happ which passes through zero at 17°C) and the presence of a secondary binding event, with a large exothermic effect, after saturation of the primary binding site (Fig. 2). It was concluded that measurement of the entire binding isotherm was not possible by ITC. However, conditions could be chosen that were optimal for the direct determination of ∆Happ by ITC (i.e., total association at partial saturation (see Subheading 1.2.). At the chosen DNA concentration of 60 µM, total association of injected Sox-5 in an ITC experiment is achieved (the ratio of the DNA concentration to the estimated KD being approx 1700). The fractional molar saturation was always kept lower than 0.5 to avoid any secondary binding effects. Because high concentrations were to be used, the total heat effect was greater and more easily detectable, which meant that ∆Happ values could be accurately obtained at temperatures close to 17°C. Additional experiments included low-speed analytical ultracentrifugation at different temperatures to demonstrate that the free Sox-5 is monomeric. For the free DNA, UV melting was performed as an initial check on its melting temperature and for two-state melting of the duplexes. UV melting of the


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Fig. 2. ITC titration experiment of Sox-5 into a 12-bp DNA duplex. (A) Fifteen injections of 540 µM Sox-5 into 10 µM of 12-bp DNA at 9°C. The injection rate was 5 µL in 8 s, with a time interval of 5 min between injections. (B) a plot of ∆Happ against ratio of Sox-5 to DNA showing the primary endothermic and secondary exothermic reactions that occur on either side of the stoichiometric point.

Sox-5–DNA complex showed a single cooperative transition with a melting temperature above that of the free DNA.

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2. Materials 2.1. Calorimeters Isothermal titration calorimetry was performed on an OMEGA or MCS-ITC titration calorimeter (MicroCal Inc., Northampton, MA). Technical details on the construction of mixing microcalorimeters, their performance and sensitivity, and on the theory of data analysis have been described elsewhere (17–20). Differential scanning calorimetric experiments were usually carried out on a Nano-DSC calorimeter (Calorimetric Science Corp., Utah). The instrument’s performance and data acquisition are detailed in ref. 21. In brief, the instrument operates over the temperature range –20°C to 130°C at any set heating/ cooling rate. The sample and reference calorimetric cells are of approx 900 µL volume. Control of the calorimeter and scan acquisition is achieved via the supplied DSC_Acquisition program running on a PC computer connected to the calorimeter. In experiments in which the amount of sample was limiting, a new version of the Nano-DSC calorimeter having cells of approx 300 µL volume was used.

2.2. Reagents and Solutions 2.2.1. Calorimetry 1. For calorimetric experiments, it is important to ensure that the protein and DNA samples are homogeneous, because even a few percent of contaminating species might cause significant heat effects. Homogeneity of the samples is most reliably verified by mass spectrometry. 2. Concentrations must be accurately determined because this affects the DSC and ITC data (see Subheading 3.4.). The preparation of equimolar mixtures (of complementary oligonucleotides or Sox-5-DNA complex) can then be made precisely, without further purification. Dilutions are made by weight on a precision balance. 3. The DSC and ITC experiments with the Sox-5–DNA complex used a working buffer of 100 mM KCl, 10 mM potassium phosphate, and 1 mM EDTA (pH 6.0) (see Note 1). All buffer solutions were prepared from the highest-quality reagents and ultrapure water. For ITC experiments, the solutions were filtered through a 0.45-µm membrane and thoroughly degassed prior to use.

2.2.2. Preparation of Sox-5 HMG Box–DNA Complexes and Components 1. DNA oligonucleotides were synthesized using phosphoramidite chemistry and purified on a Mono Q HR16/10 column fitted to a Pharmacia FPLC system, eluting with a linear 0.1 M to 1.0 M NaCl gradient in 10 mM Tris-HCl, 1 mM EDTA, and 20% (v/v) acetonitrile (pH 7.0). Fractions containing oligonucleotide were precipitated with 3 vol of ethanol at –20°C overnight, then centrifuged down and


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redissolved in water. Oligonucleotides were extensively dialyzed using Spectrapor tubing (molecular-weight cutoff 500 Daltons) against three changes of 1 L working buffer at 4°C. 2. DNA duplexes were prepared by mixing equimolar amounts of the complementary oligonucleotides in working buffer and annealed by heating to 95°C in a water-bath followed by slow cooling to 4°C over a period of approx 4 h. DNA duplexes were then extensively dialyzed using 3000-Dalton molecular-weight cutoff tubing against three changes of 1 L working buffer at 4°C. 3. The HMG box of mouse Sox-5 (amino acids 182 to 260, [15]) was expressed as a fusion protein in pGEX-2T, using E. coli BL21 (DE3) plysS cells. After affinity purification with glutathione-agarose and thrombin cleavage while still attached to the column (22), reverse-phase high-performance liquid chromatography (HPLC) was used to purify the protein. Protein was redissolved in water and refolded by extensive dialysis against three changes of 1 L working buffer at 4°C. 4. The Sox-5–DNA complex was prepared by the mixing together of equal volumes of the components (at equimolar concentration) in working buffer at 4°C. Sox-5 was added in 10% aliquots, at 5-min intervals, to the DNA. The complex was then extensively dialyzed using 3000-Dalton molecular-weight cutoff tubing against three changes of 1 L working buffer at 4°C. The accuracy of the concentrations of protein and DNA used to form the 1:1 complex may be checked by trial additions of Sox-5 to DNA at various protein:DNA ratios followed by electrophoresis on a nondenaturing polyacrylamide gel.

2.2.3. DNA Concentrations 1. 100 mM Tris-HCl (pH 8.0). 2. Snake venom phosphodiesterase I (PDE1, from Crotalus durissus terrificus, Sigma).

3. Methods 3.1. Isothermal Titration Calorimetry Before a series of experiments, the calorimeter was calibrated either by applying electrically generated heat pulses or by standardized chemical reactions (e.g., the protonation of tris[hydroxymethyl] aminomethane or the binding of Ba2+ to 18-crown-6 ether). It is recommended to equilibrate the jacket with a circulating water-bath for about 10 h. at a temperature lower than the temperature of the experiment by 3–5°C. Although such equilibration is not strictly necessary when operating at above room temperature, it substantially improves the baseline stability. The stirring speed during both the equilibration and the experiment was 350 rpm (see Note 2). 1. Sample and reference cells are first rinsed with dialysis buffer. The reference cell is then filled with buffer and the sample cell filled with the DNA solution. The system is heated to the working temperature and equilibrated until the differen-

522

2.

3.

4.

5.

Read and Jelesarov tial power signal levels off. The injection syringe containing Sox-5 is inserted into the sample cell, stirring is initiated, and the baseline is established over a period of 30–60 min. Typically, a baseline drift (differential power signal drift) of less than 20–30 nJ/min and an rms noise of less than 15–20 nJ/s (or nW) indicates complete thermal equilibration of the system under stirring (see Note 3). The experiment is started with a small injection of 1–2 µL. The reason for this is that during the long equilibration period, diffusion through the injection ports occurs, thus causing a change in protein concentration near the syringe needle tip. The actual injection schedule is then executed. To measure the enthalpy of Sox-5 binding to DNA, six to eight injections each of 8–12 µL and of 12–15 s duration were performed, with a 5-min interval between injections (see Note 4). Typical thermograms are depicted in Fig. 3A, traces a and c. After completion of the experiment, the cells are thoroughly cleaned. Cleaning of the calorimetric cells and filling syringes follows a standard laboratory protocol (see Note 5). The cells may then be filled with buffer and step 2 repeated. Injections of protein into buffer will yield the heat associated with protein dilution and other nonspecific effects. Typical control thermograms are depicted in Fig. 3A, traces b and d. Because the heats obtained in steps 2 and 4 are directly compared in the data analysis, it is crucial that (1) the blank titration is performed at exactly the same temperature as the main experiment, (2) the same protein solution and dialysis buffer are used, and (3) an identical injection scheme is executed. If any of the listed requirements is not fulfilled, the results of the experiment could be misleading. The titrations are performed at different temperatures to collect data on the temperature dependence of the binding enthalpy.

3.1.1. ITC Data Analysis If a complete binding isotherm has been recorded over an optimal concentration range, the data can be subjected to a nonlinear least-squares analysis to obtain a full set of parameters (∆Happ, KA, and n) according to Eq. 4. In the case of Sox-5 binding to DNA, the experiments were designed to measure only the enthalpy and heat capacity changes. Thus integration of the differential power peaks (see Note 6) collected as in step 2 of Subheading 3.1. yields ∆qi,app and the nonspecific heats ∆qi,ns are obtained by integration of the peaks collected as in step 4 of Subheading 3.1. Under conditions of total association ∆[P]i,bound equals ∆[P]i,total and is easily calculated from the known concentration of protein in the injection syringe, the volume of each injection and the Fig. 3. (opposite page) Representative records of a Sox-5 titration into DNA duplex (A) and enthalpies of binding of Sox-5 to a 12-bp DNA duplex as measured by ITC (B). (A) Trace a: six injections of 550 µM Sox-5 into 58 µM of 16-bp DNA at 9°C (i.e., up to a fractional saturation of 0.45); trace b: control titration of Sox-5 into buffer at


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9°C; trace c: eight injections of 490 µM Sox-5 into 60 µM of 12-bp DNA at 30°C; trace d: control titration of Sox-5 into buffer at 30°C. The injection rate was 8 µL in 12 s. The time intervals between injections were 4 min (traces a and b) and 5 min (traces c and d). (B) The open circles represent ∆Happ measured between 9 and 30°C and calculated according to Eq. 6 (mean values of six to eight injections). Note that ∆Happ changes sign at about 17°C. The standard deviations from the mean values shown are of the order of ±5–10%. Filled circles represent the binding enthalpy obtained after correction of ∆Happ for protein and DNA refolding and complex unfolding. The lines correspond to linear least-squares fits to the data, the slope of which is equal to ∆Cp.

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volume of the cell, Vcell. The enthalpy of association, ∆Happ, at temperature T may then be calculated from Eq. 6. This procedure is repeated for the pairs of titrations at different temperatures. The experimentally observed enthalpies (∆Happ) obtained at a number of temperatures can be plotted as illustrated in Fig. 3B.

3.2. Differential Scanning Calorimetry 1. Prior to starting experiments ensure that the Nano-DSC calorimeter sample and reference cells are at room temperature, thoroughly cleaned, and thermally balanced. 2. Add working buffer (100 mM KCl, 10 mM potassium phosphate, and 1 mM EDTA (pH 6.0)) which is at room temperature to both sample and reference cells. It is most important that no air bubbles are present in the calorimeter cells (see Note 7). The presence of air bubbles will yield incorrect values for the partial molar heat capacity of the macromolecule, because the volume of solution in the sample and reference cells is not identical (assumed in Eq. 7). Furthermore expansion of the minutes of air bubbles with heating results in significant heat output (and vice versa on cooling). 3. Seal the top of the chamber with the screw-threaded piston and let the calorimeter settle to thermal equilibrium. Apply approx 2 atm of overpressure to the cells by screwing down the piston. As pressure is applied note the compensatory power reading—it should not change by more than a few microwatts (see Note 8). 4. Scan to obtain a buffer–buffer baseline. Initially, a number of scans are recorded in order to obtain a number of reproducible baselines (to within 0.5 µW, in the linear region). A complete cycle of heating and cooling at a rate of 1 K/min actually takes about 4 h because at the end of each heating or cooling scan, there is a period of thermal equilibration. 5. Remove the buffer from the sample cell. This is most easily accomplished with a vacuum line attached to a water pump. 6. Slowly and carefully apply a 1.8-mL dialyzed sample to the cell, using the method outlined in Note 7. For concentrated protein samples, care is needed to remove all air bubbles. The excess sample is put to good use: first to determine sample concentration and, second, it is diluted with the final dialysate for use in further calorimetric runs. Again, apply a pressure of approx 2 atm to the cells. 7. Perform one heating and cooling scan to obtain the denaturation and renaturation curves of the sample-buffer. Usually samples were heated from 0°C to 80°C and then cooled to –8°C, both at a rate of 1 K/min. The heat effects of unfolding and refolding should appear as mirror images, slightly shifted in temperature because the slower kinetics of refolding. Reproducibility shows that the heat-induced unfolding/refolding is reversible. However, this may not be the case (see Note 9). 8. Remove the sample from the cell. Thoroughly wash out the cell with buffer and then perform another scan of buffer. A heat-asorption peak may be observable if the previous sample is not completely removed (see Note 10).


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3.2.1. DSC Data Analysis The subtraction of buffer scans to obtain an accurate baseline, the conversion to partial molar heat capacities, and the deconvolution of the excess molar heat capacity function into separate transitions were all performed using the CPCalc program. CPCalc provides a simple interactive mechanism, based on Data Exchange Ports, for the parsing of data from one step to another. 1. The functions of differential power and temperature vs time are extracted from the DSC data acquisition files. Each file may contain more than one scan, but further analysis is on a one-scan basis. 2. A matching buffer–buffer scan is subtracted from a sample-buffer scan. Both must be from a heating (or cooling) scan. Since the absolute values of the molar heat capacities depend on good baseline subtraction and the buffer-buffer scans vary to a small extent, it is important that a number of subtractions are tried using different scans from the acquisition file. 3. The subtracted compensatory power curve is converted to the partial molar heat capacity function by use of Eq. 7. Accurate values for the concentration (Subheading 3.4.), molecular mass, and partial specific volume of the macromolecule are required (see Note 11). 4. Appropriate functions for the intrinsic heat capacity of the initial and final states may then be put onto the partial molar heat capacity function (Fig. 4). The calorimetric ∆H, ∆S, and melting temperature of the peak are obtained. Enthalpies and entropies over a restricted temperature interval, ∆H(T) and ∆S(T), are obtained by viewing the partial molar heat capacity function between the two temperatures. 5. The excess molar heat capacity function is deconvoluted into separate transitions.

3.3. Correction of ITC Derived Enthalpies The value for ∆C p,app calculated from the slope of the ∆Happ vs T plot (Fig. 3) is about –3.3 kJ/K/mol, implying that a considerable amount of hydrophobic surface is buried at the complex interface. However, over the temperature range (10–30°C) used for the ITC measurements, the partial molar heat capacity functions for both free Sox-5 and DNA (DSC data, Fig. 4A) are greater than the calculated intrinsic heat capacity functions for the fully folded molecules. Thus, the DSC experiments show significant heat absorption resulting from a temperature-dependent unfolding of the free Sox-5 and DNA. Significant refolding of both components must therefore take place when they associate at these temperatures: a simple interaction between rigid-body molecules is not observed. If one wishes to relate observed energetic parameters to structural features of a rigid fully-folded complex, then the enthalpic contributions arising from refolding of the free Sox-5 and DNA, as determined from DSC, must be added to the ITC-measured enthalpies of association to obtain the enthalpies applicable to a rigid-body interaction. Furthermore, because the

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Fig. 4. (A) Partial molar heat capacity functions obtained for the free Sox-5 protein (dotted curve), the 12-bp DNA duplex that includes the AACAAT motif (dot–dash curve) and the 1:1 Sox-5–12-bp DNA complex (solid curve). Note that the complex dissociates in a single cooperative trimolecular transition with a melting temperature only slightly above that of the 12-bp DNA duplex. The free Sox-5 protein and 12-bp DNA duplex dissociate in a monomolecular and bimolecular manner, respectively. (B) Partial molar heat capacity function for the Sox-5–12-bp DNA complex showing the intrinsic heat capacity functions for the fully folded (N) and unfolded (D) complex (thin lines). The area above the dashed line corresponds to the total enthalpy of all the temperature-induced changes in the complex.


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complex also absorbs heat over the 10–30°C temperature range (i.e., becomes partially unfolded [Fig. 4A]), such heats must be subtracted from the ITCmeasured enthalpies of association to generate the net enthalpies applicable to the formation of a fully folded complex. These corrections considerably affect the enthalpy profile of binding (see Fig. 3B). The corrected plots of ∆Happ vs T are now linear, with an average slope of about –1.5 kJ/K/mol and this corrected value of ∆Cp is in very good agreement with the amount and type of molecular surface screened from the solvent upon association, calculated assuming a rigid-body interaction.

3.4. Determination of Concentrations The protein and DNA samples must be highly homogenous and their concentrations must be known to the highest possible precision. In the first place this enables an accurate preparation of DNA duplex from its complementary oligonucleotides and of the Sox-5–DNA complex from protein and DNA duplex. In DSC, the accuracy with which the sample concentration can be determined directly affects the molar heat capacity values obtained. In ITC, unlike other binding assays, errors in the concentration of protein are directly reflected in the values of ∆Happ (Eqs. 3 and 4). Thus, in both ITC and DSC, concentrations are determined after dialysis. In the DSC experiments, it is also possible that a small amount of buffer from a previous scan remains in the gold capillary cell. To eliminate this potential source of error, concentrations were determined using the excess sample that remains after filling the calorimeter cell.

3.4.1. DNA For oligonucleotides and DNA duplexes, concentrations were determined from their UV absorption at 260 nm, after digestion to nucleotides with snake venom phosphodiesterase I (PDE1). 1. Accurately dilute the solution of DNA to about 1.0 mL with 100 mM Tris-HCl (pH 8.0) so as to give an absorbance at 260 nm of about 0.5 in a 1-cm pathlength cell. To overcome any reliance on the presumed accuracy of the pipets being used, the dilutions are performed on a precision balance. 2. Record the UV absorption spectrum of the diluted DNA solution, using as reference a blank cell containing buffer. Note that for small oligonucleotides and DNA duplexes with a biased nucleotide composition, the absorption maximum is not necessarily at 260 nm. This is in fact an optional step, but the recording of UV spectra both before and after the addition of PDE1 indicates that digestion has occurred and enables a determination of the hypochromicity of the DNA solution. 3. Remove the diluted DNA solution from the cell and place into a screw-capped 1.5-mL tube. Add either 0.008 U (for oligonucleotides) or 0.08 U (for DNA

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duplexes) of PDE1 to the solution, mix well, and incubate at 37°C overnight. If necessary, a time-course of the digestion may be followed (at room temperature) in the UV spectrophotometer by direct addition of PDE1 to the DNA solution in the quartz cell. 4. Accurately record the UV absorption spectrum of the DNA solution after PDE1 digestion, using a black cell containing buffer as a reference. The contribution to the absorbance at 260 nm arising from overlap of the PDE1 280-nm peak may be neglected 5. Optionally, calculate the percent hypochromicity (%H) from the equation %H =

(Aa – Ab) × 100 Aa

where Ab and Aa are the absorbances at 260 nm before and after PDE1 digestion respectively, with dilution the result of the addition of PDE1 taken into account. Values of %H are usually approx 20% for oligonucleotides and approx 35–40% for duplex DNA. Lower values indicate that digestion may not be complete, that the original DNA is degraded, or that there is a failure to form duplex DNA. 6. Calculate the molar nucleotide concentration and thus the molar DNA concentration from the following equations [Nucleotide] =

Aa(Dilution)N (12,010G) + (15,200A) + (8400T) + 7050C)

and [DNA] =

[Nucleotide] N

where N is the total number of nucleotides in the DNA and G, A, T, and C are respectively the number of dG, dA, dT, and dC nucleotides. The extinction coefficients at 260 nm for the four nucleotides are from ref. 23.

3.4.2. Protein The concentration of Sox-5 (in working buffer) was determined from its UV absorption at 280 nm using an extinction coefficient of 17,460/M/cm. This extinction coefficient is based on the addition of the extinction coefficients of tryptophan and tyrosine (of which there are two and five in Sox-5, respectively). The accuracy of the concentration determination is within 5%.

3.4.3. Sox-5–DNA Complex The concentration of complex was determined from its UV absorbance at 260 nm. At this wavelength, the absorption is mainly the result of the DNA, but there is a significant absorption from the protein that must be taken into account. There may also be additional hyperchomic effects because the DNA of the Sox-5–DNA complex is highly bent with considerable base unstacking.


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Thus, the approach taken was to measure the UV absorbance at 260 nm for a solution of DNA duplex and then again after the addition of an equimolar amount of Sox-5 protein to form the complex. The measurement was performed at approx 60 µM DNA in a 1-mm pathlength cell, such that the complex was fully associated. For the Sox-5–12-bp DNA complex, an extinction coefficient at 260 nm of 158.6 × 103/Molar complex/cm was obtained. 4. Notes 1. The choice of assay buffer depends on the system under study and on the requirements of other assays performed in the context of a particular investigation. Buffers with a different heat of protonation may be used in an ITC experiment to check whether the protonation state of the system changes upon association. 2. In setting the stirring speed, the particular system under study must be considered. Some proteins do not resist the forces arising from rapid stirring of the solution placed in the narrow calorimetric cell and aggregate or unfold. On the other hand, very slow stirring may result in low rates of heat transfer, thus causing broadening of the peaks observed in the thermogram and a decrease in sensitivity. 3. When working below room temperature, it is recommended to fill the cells with cold solutions, otherwise the equilibration time may be very long. Any particles in the stirred sample cell as well as the formation of bubbles during filling may cause problems in establishing the baseline. 4. There is no general rule about the number, volume, and duration of injections. To obtain an entire binding isotherm a 2–4 molar excess of ligand over the concentration of receptor binding sites should be injected by at least 10–15 additions. Five to 10 injections suffice to reliably measure the enthalpy of reaction. In this case, the degree of saturation must be less than 0.5 at the end of experiment. The volume and duration of injections should be chosen in such a way that the released heat is well above the threshold of sensitivity and sharp peaks appear in the thermogram. Typically, 5-min intervals between injection are sufficiently long times for the signal to return to the baseline. However, for very slow reactions, this interval should be prolonged. Because the heat of reaction is a temperaturedependent quantity, the injection scheme might be changed for experiments carried out at different temperatures. 5. Alternating cycles of washing with 0.5 M NaOH and isopropanol are routinely used. To remove heavily precipitated proteins, the cell can be rinsed with a hot solution of 20% sodium dodecyl sulfate (SDS). The protocol for cleaning will depend on the particular system under study and the material of the cell. 6. Integration requires construction of a proper baseline. Some software products support automatic procedures for baseline determination. However, it is often found that manual adjustment of the baseline is a better practice, particularly in cases when the signal-to-noise ratio is low or when the instrument baseline drift is substantial. In constructing the baseline manually, it is important to use the

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Fig. 5. Arrangement for filling the capillary cells in the Nano-DSC calorimeter. same integration window for all the peaks observed, both in the specific binding and in the blank titration experiments. This will avoid the introduction of nonrandom bias in the data. 7. We would advocate filling each cell using a minimum of approx 1.8 mL of solution by the following method (see also Fig. 5). First attach a 1-mL pipet tip using a short length of silicon tubing to one port of the cell. Draw up 0.9 mL of solution using a pipettor fitted with a 1-mL tip and silicon tubing. Connect this to the other side of the cell and slowly introduce the solution into the cell. Disconnect and apply a second 0.9 mL of solution, without introducing any air bubbles. As the cell is only about 0.9 mL in volume, excess solution rises up into the 1-mL


8.

9.

10.

11.

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pipet tip attached to the other side. The solution is then pipetted up and down, at first slowly and then more rapidly, for sufficient time to expel minute air bubbles from the cell. Eventually, excess solution may be removed by carefully withdrawing it into the 1.0-mL tip connected to the pipettor and then disconnecting by simultaneously pulling off both 1-mL tips with their connecting tubing. If the microwatt reading rapidly increases or decreases on applying pressure, then air bubbles are present in either of the cells. The cells must then be emptied and refilled. One reason may be temperature-induced aggregation, manifesting itself as a large peak of heat evolution that typically commences close to the peak of maximum heat absorption of the cooperative transition (approximately at the melting temperature, Tm). A s possible reason for irreversibility is the presence of disulfide bonds in the protein, which may disrupt on heating but, on cooling form, mixed disulfide bonds with incorrect Cys residues. This may be overcome by using proteins (or domains) with no Cys residues (as in Sox-5) or by using engineered versions in which the Cys residues are mutated to Ser. This strategy will, however, depend on the contribution of the disulfide bonds in stabilizing the overall protein fold. The calorimetric cells may be thoroughly cleaned by filling them with 50% (v/v) formic acid, followed by heating from 25°C to 65°C (at 1 K/min). The cells are then thoroughly rinsed with water and buffer. Multiple baseline scans of the buffer are recommended before proceeding with the next sample. The partial specific volumes used for oligonucleotides and DNA duplexes were 0.53 and 0.54 mL/g. The partial specific volume of Sox-5 was calculated from its composition as 0.723 mL/g, using the known partial specific volumes of the amino acids. The Sox-5–12-bp DNA complex partial specific volume of 0.650 mL/g was calculated as the weight average of the partial specific volumes of Sox-5 and the DNA duplex.

Acknowledgments We would like to thank Peter Privalov (Johns Hopkins University, Baltimore), in whose laboratory the calorimetry was performed. We thank Colyn Crane-Robinson for critical reading of the manuscript. Financial support from an NIH grant to the Baltimore laboratory (GM48036-06), a Wellcome Trust grant to the Portsmouth laboratory, and NATO Collaborative Research Grants are gratefully acknowledged. References 1. Patikoglou, G. and Burley, S. K. (1997) Eukaryotic transcription factor–DNA complexes. Ann. Rev. Biophys. Biomol. Struct. 26, 289–325. 2. Schultz, S. C., Shields, G. C., and Steitz, T. A. (1991) Crystal structure of a CAP– DNA complex: the DNA is bent by 90°. Science 253, 1001–1007. 3. Kim, J. L., Nikolov, D. B., and Burley, S. K. (1993) Co-crystal structure of TBP recognising the minor groove of a TATA element. Nature 365, 520–527.

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4. Werner, M. H., Huth, J. R., Gronenborn, A. M., and Clore, G. M. (1995) Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY–DNA complex. Cell 81, 705–714. 5. Love, J. J., Li, X., Case, D. A., Giese, K., Grosschedl, R., and Wright, P. E. (1995) Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature 376, 791–795. 6. Privalov, P. L., Jelesarov, I., Read, C. M., Crane-Robinson, C., et al. (1999) The energetics of HMG box interactions with DNA. Thermodynamics of the DNA binding of the HMG box from mouse Sox-5. J. Mol. Biol. 294, 997–1013. 7. Crane-Robinson, C., Read, C. M., Cary, P. D., Driscoll, P. C., Dragan, A. I., and Privalov, P. L. (1998) The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5. J. Mol. Biol. 281, 705–717. 8. Jelesarov, I., Crane-Robinson, C., and Privalov, P. L. (1990) The energetics of HMG box interactions with DNA. Thermodynamic description of DNA duplexes the target. J. Mol. Biol. 294, 981–995. 9. Murphy, K. P. and Freire, E. (1992) Thermodynamics of structural stability and co-operative folding behaviour in proteins. Adv. Protein Chem. 43, 313–361. 10. Spolar, R. S., Livingstone, J. R., and Record, M. T., Jr. (1992) Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water. Biochemistry 31, 3947–3955. 11. Ferrari, M. E. and Lohman, T. M. (1994) Apparent heat capacity change accompanying a nonspecific protein-DNA interaction. Escherichia coli SSB tetramer binding to oligodeoxyadenylates. Biochemistry 33, 12,896–12,910. 12. Bruzzese, F. J. and Connelly, P. R. (1997) Allosteric properties of inosine monophosphate dehydrogenase revealed through the thermodynamics of binding of inosine 5'-monophosphate and mycophenolic acid. Temperature dependent heat capacity of binding as a signature of ligand-coupled conformational equilibria. Biochemistry 36, 10,428–10,438. 13. Privalov, P. L. and Makhatadze, G. I. (1990) Heat capacity of proteins. J. Mol. Biol. 213, 385–391. 14. Makhatadze, G. I. and Privalov, P. L. (1995) Energetics of protein structure. Adv. Protein Chem. 47, 307–325. 15. Denny, P., Swift, S., Connor, F., and Ashworth, A. (1992) A Sry-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA-binding protein. EMBO J. 11, 3705–3712. 16. Connor, F., Cary, P. D., Read, C. M., Preston, N. S., Driscoll, P. C., Denny, P., et al. (1994) DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5. Nucleic Acids Res. 22, 3339–3346. 17. McKinnon, I. R., Fall, L., Parody-Morreale, A., and Gill, S. J. (1984) A twin titration microcalorimeter for the study of biochemical reactions. Anal. Biochem. 139, 134–139. 18. Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137.


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19. Freire, E., Mayorga, O. L., and Straume, M. (1990) Isothermal titration. Anal. Chem. 62, 950A–959A. 20. Breslauer, K. J., Freire, E., and Straume, M. (1992) Calorimetry: a tool for DNA and ligand–DNA studies. Methods Enzymol. 211, 533–567. 21. Privalov, G., Kavina, V., Freire, E., and Privalov, P. L. (1995) Precise scanning calorimeter for studying thermal properties of biological macromolecules in dilute solution. Anal. Biochem. 232, 79–85. 22. Read, C. M., Cary, P. D., Preston, N. S., Lnenicek-Allen, M., and Crane-Robinson, C. (1994) The DNA sequence specificity of HMG boxes lies in the minor wing of the structure. EMBO J. 13, 5639–5646. 23. Wallace, R. B. and Miyada, C. G. (1987) Oligonucleotide probes for the screening of recombinant DNA libraries. Methods Enzymol. 152, 432–442.

Surface Plasmon Resonance

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36 Surface Plasmon Resonance Applied to DNA–Protein Complexes Malcolm Buckle 1. Introduction 1.1. The Relationship Between Refractive Index and Mass Surface plasmon resonance (SPR) measures refractive index changes (∆n) at or near a surface and relates these to changes in mass at the surface (Fig 1). This relationship is given by the Clausius Mossotti form (Eq. 2) of the Debye equation (Eq. 1): ε–1 N α+µ = ε + 2 3ε0 kT

(1)

ε–1 Nα = ε+2 3ε0

(2)

( )

where ε is the real part of the dielectric constant or permittivity constant related to the refractive index by ε = n2, N is the number density given by NAρ/Ma (NA is Avogadro’s number, ρ is the density and Ma is the molecular mass). It is assumed that ∆n/∆C is a constant.

1.2. SPR Using the BIACORE Instrument In physical terms, the detection system of this SPR machine consists of a monochromatic, plane polarized light source, and a photodetector that are connected optically through a glass prism (Fig. 1). A thin gold film (50 nm thick), deposited on one side of the prism, is in contact with the sample solution. This gold film is, in turn, covered with a long-chain hydroxyalkanethiol, which forms a monolayer (approx 100 nm thick) at the surface. This layer essentially serves as an attachment point for carboxymethylated dextran chains that create a hydrophilic surface to which ligands can be covalently coupled. Light From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Buckle

Fig. 1. Schema showing the principle of surface plasmon resonance. Light from a laser source arriving through a prism at a gold surface at the angle of total internal reflection (θ) induces a nonpropagative evanescent wave that penetrates into the flow cell opposite the prism. The intensity of the reflected light is continuously monitored. At a given angle (λ) dependent on the refractive index of the solution in the flow cell, resonance between the evanescent wave and free electrons in the gold layer results in a reduction in the intensity of reflected light. The change in angle of reduced intensity (∆λ) reflects changes in the refractive index (n) of the solution in the flow cell immediately adjacent to the gold layer. A dextran surface coupled to the gold layer allows immobilization of ligands (e.g., DNA) within the evanescent field. incident to the back side of the metal film is totally internally reflected onto the diode-array detector. A property of this situation is that a nonpropagative evanescent wave penetrates into the solution side of the prism away from the light source. Free electrons in the gold layer enter into resonance with the evanescent wave. In fact, such resonance implies that the amplitude vector character→ p) is equal to izing a transversal wave propagating along the gold surface (ks → 2 the component (kx ) of the evanescent wave. Because ε = n , if ω is the frequency of the wave and c the speed of light, then

ksp =

ω c

ε1 − ε 2 ε1 + ε 2

(3)

furthermore, given that for the evanescent wave,

kx =

ω sin θ εg c

(4)


537

| | | |

when resonance occurs, ksp = kx and the intensity of the reflected light decreases at a sharply defined angle of incidence, the SPR angle, given by the simple expression

sin·θ 0 =

ε 1ε 2 εg(ε 1 + ε 2)

(5)

Thus, θ0, the SPR angle at which a decrease in the intensity of reflected light occurs, measures the refractive index of the solution in contact with the gold surface and is dependent on several instrumental parameters (e.g., the wavelength of the light source and the metal of the film). When these parameters are kept constant, the SPR angle shifts are dependent only on changes in refractive index of a thin layer adjacent to the metal surface. Any increase of material at the surface will cause a successive increase of the SPR angle, which is detected as a shift of the position of the light intensity minimum on the diode array. This change can be monitored over time, thus allowing changes in local concentration to be accurately followed. The SPR angle shifts obtained from different proteins in solution have been correlated to surface concentrations determined from radio-labeling techniques and found to be linear over a wide range of surface concentration. The instrument output, the resonance signal, is indicated in resonance units (RU); 1000 RU correspond to a 0.1° shift in the SPR angle, and for an average protein, this corresponds to a surface concentration change of about 1 ng/mm 2 (for nucleic acids, see Note 1). It is remarkable that the present instrument (Biacore 2000) can measure a deviation of 10–3°, in other words, a variation of 10–5 in the refractive index .

1.3. Immobilization of DNA to a Surface Although a variety of techniques exist for the immobilization of DNA on the dextran surface, the most efficient for the majority of protein–DNA interactions is the use of immobilized streptavidin that can then interact with a suitably end-labeled DNA molecule. The streptavidin is immobilized via a carbodiimide–N-hydroxyl succinimide coupling reaction to the carboxyl groups of the dextran (Fig. 2). DNA is easily obtained either by direct purchase of oligomers end-labeled with biotin, or, for larger fragments, direct polymerase chain reaction (PCR) from biotinylated oligomers. Unless a particularly unusual configuration is required, biotin is generally present at one end of the DNA molecule and on one strand if the DNA is double stranded. The end biotinylated DNA is then flowed across the surface and allowed to bind to the desired final concentration (Fig. 3).

1.4. Protein Binding to Immobilized DNA The protein is flowed across the immobilized DNA in a buffer and at a temperature suitable for the interaction being studied (Fig. 4). A range of concen-

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Buckle

Fig. 2. Coupling of streptavidin to a flow cell activated by carbodiimide and hydroxyl succinimide. The figure shows a sensorgram of a carboxymethylated dextran surface (CM5) activated by carbodiimide and N-hydroxylsuccinimide prior to coupling with streptavidin. Sharp changes in the resonance units (RU) reflect bulk refractive index changes as a result of differences in the buffer. Ethanolamine is used to block all unreacted activated carboxyl groups. The difference between the final RU value and the initial value presents an accurate measure of the amount of streptavidin covalently coupled to the surface.

trations should be investigated. A flow rate in excess of 10 µL/min is advised and a sufficient contact time during the association phase to saturate the immobilized DNA (as seen by a steady-state plateau for the RU values) at protein concentrations in excess of the anticipated Kd for the interaction. The dissociation phase should also be allowed to continue for at least sufficient time to allow over a third of the complex to dissociate.

1.5. Binding Curve Analysis 1.5.1. Stoichiometry and Equilibrium Analysis For an immobilized DNA fragment (D), the interaction with a mobile protein (P) can be written as ka

D + P ↔ DP

(Scheme 1)

kd

A classical Langmuir adsorption isotherm requires that the fraction of available sites on the DNA occupied by the protein (θD) be given by


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Fig. 3. Immobilization of a biotinylated DNA fragment to a streptavidin surface. In this example, a 200-bp fragment of DNA (10 µg/mL) containing a single biotin label at one 5' end was flowed at 20 µL/min in HBS buffer over the streptavidin surface. The initial bulk refractive index change was followed by a gradual increase reflecting DNA binding to the streptavidin. At the end of the DNA injection phase, the bulk refractive index change was recovered and the difference in absolute RU values compared to the initial value reflects the number of molecules of DNA now bound to the surface.

θD =

DP Dt

(6)

Furthermore, in such a simple case, the equilibrium association constant Ka is given by the expression θD =

KaP 1 + KaP

(7)

Thus, in an SPR experiment, the steady-state level of bound protein at a given concentration of total protein should be calculated from the asymptote of the sensorgram and the RU values converted into moles of bound protein. Assuming that in the continuous-flow system typical of Biacore SPR machines, [P]T = [P], a plot of θD against [P]T should allow a direct fit by Eq. 7 to give an estimation of Ka, from which we obtain Kd = 1/Ka.

1.5.2. Kinetic Analysis The protein that is injected across the surface should after an infinite time arrive at an association equilibrium giving a signal Req, and the resonance signal R at time t during this process following injection at t = 0 when R = R0, should, in simple instances, obey the expression

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Buckle

Fig. 4. Protein binding to immobilized DNA. In this example, purified RNA polymerase (120 nM) from Eschericia coli was injected at 20 µL/min across an immobilized 203-bp DNA fragment containing a promoter sequence (continuous line [a]). The dotted line (b) shows the same protein flowing across a streptavidin surface without immobilized DNA. Note the large bulk refractive index effects resulting principally from the presence of glycerol in the protein solution and nonspecific binding. This is a complex phenomenon composed not only of electrostatic interactions with the dextran but also necessary transient interactions with nonpromoter DNA. The dissociation phase is characterized by a steady decrease in signal lending itself to the type of analysis described in the text. The association phase is more complicated. In this instance an example of how the association phase may be dealt with is given in ref. 7. Rt = R0 – (Req – R0) (1 – e–kobst )

(8)

Similarly, for the dissociation of the bound protein, Rt = R0 + (Req – R0) (e–kofft)

(9)

assuming that the bound molecule completely dissociates from the immobilized ligand. Consequently, the observed reaction rate kobs for the interaction is given by kobs + kon[P] + koff

(10)

There is thus a linear relationship between the value for kobs and the total concentration of protein [P]. The value for kobs can be obtained from a direct fit of the association phase using Eq. 8, or by linear regression of a semi-log plot. It thus follows that linear regression analysis of the dependence of kobs on [P] allows the calculation of kon and koff using Eq. 7. If we assume that the reaction is in fact activation controlled (were it otherwise, then the association rate


541

would be of the order of 109/M/s, which is well beyond the range of current SPR devices), then koff = konKd

(11)

Thus, the equilibrium dissociation constant (Kd) can be obtained from the ratio of the off and on rates. There are many pitfalls to using SPR, which are covered in several fairly recent reviews (2,3). In the case of the Biacore instrumentation, the new data evaluation software deals with certain situations. What it cannot do is to determine the best strategy for setting up an experiment. In summary, certain important points must be taken into account even in the simple analysis given above. Several of these are covered in Notes 2–6. 2. Materials 1. An SPR device. In this chapter, a Biacore instrument is referred to either as the classic Biacore or the Biacore 2000. It is recommended (but not essential) that the machine be modified such that the two racks into which samples are placed in the machine are separately thermostated. Rack 1 should be thermostated to 4°C; rack 2 should be thermostated to the temperature at which the interaction is to be measured. The protocols illustrated here require rack D in the first position and rack A in the second position. 2. Streptavidin from Pierce resuspended in 0.22 µm filtered distilled water to a final concentration of 5 mg/mL. This preparation may be stored at 4°C for up to 3 mo. 3. HBS buffer: 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% Biacore surfactant. 4. N-Ethyl-N'-(diethylaminopropyl) carbodiimide (EDC) and N-hydroxyl succinimide (NHS) purchased from Biacore as lyophilized powders are resuspended in 0.22 µm filtered distilled water to a final concentration of 100 mM each. 5. 1 M ethanolamine hydrochloride (pH 8.5), purchased from Biacore, stored at 4°C. 6. HBS buffer: 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20. 7. Sensor chip surface CM5 research grade installed in the Biacore apparatus and preprimed with HBS buffer. 8. Reaction vials for the Biacore (small, plastic = 7 mm; medium, glass = 16 mm; large, glass = 2 mL) purchased from Biacore. 9. End biotinylated DNA suspended in HBS buffer to 10 µg/mL. This DNA can either be purchased directly or constructed by polymerase chain reaction (PCR) using templates and an oligomer primer carrying a biotin group (purchased from Genset for example) as one of the primers. It is advisable to gel purify or high-performance liquid chromatography (HPLC) purify the DNA prior to immobilization.

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Buckle

3. Methods 3.1. Immobilization of the Ligand on the Surface

3.1.1. Coupling of Streptavidin 1. Prime the apparatus with HBS buffer. 2. The thawed EDC solution, in an Eppendorf tube with the top removed, is placed in rack 1 position a1 (r1a1). 3. The thawed NHS solution in an Eppendorf tube with the top removed, is placed in r1a2. 4. Streptavidin (5 mg/mL, 50 µL), in an Eppendorf tube with the top removed, is placed in r1a3; 2 mL of filtered (0.2 µm) distilled water is placed in a large glass vial in r2f7. 5. 1 M, sodium acetate buffer (1 mL, pH 4.5) is placed in a large glass vial in r2f3. 6. 1 M ethanolamine (200 µL) is placed in a large tube in r2f4. 7. Two small clean plastic vials are placed in r2a1 and r2a2. 8. An empty large glass vial is placed in r2f5. 9. The following method is programmed into the Biacore or Biacore 2000, checked for errors, and run. DEFINE APROG mixing FLOW TRANSFER TRANSFER MIX TRANSFER TRANSFER TRANSFER TRANSFER MIX END DEFINE APROG bind CAPTION activation FLOW * INJECT -0:20 RPOINT * INJECT -0:20 RPOINT * INJECT -0:20 RPOINT 15:00 RPOINT END MAIN FLOWCELL 1 APROG FLOWCELL 1 APROG END

(See Note 3.)

r1a1 r2a1 r1a2 r2a1 r2a1 r2f7 r2a2 r2f7 r2a2 r2f3 r2a2 r1a3 r2a2 r2a2

r2a1 EDC/NHS -b r2a2 streptavidin r2f4 ethanolamine bound

mixing bind

20 50 50 50 200 290 5 5 50

!rack1a1 = EDC !rack1a2 = NHS !rack2a1 = EDC/NHS mix !rack2f7 = distilled water !rack2f7 = distilled water !rack2f3 = 1 M acetate pH 4.5 !rack1a3 = streptavidin (5 µg/mL)

20 50 30 35

!Ethanolamine (1 M)


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3.1.2. Immobilization of the DNA 1. Place the streptavidin-activated sensor chip surface CM5 research grade in the Biacore apparatus and preprime with HBS buffer. 2. Select a surface pretreated with streptavidin. 3. Flow HBS buffer at 20 µL/min across the surface. 4. Inject the DNA solution across the surface, set the baseline to the point of injection, and monitor the change in RU during the injection phase. Ideally, between 20 and 100 RU of DNA should be immobilized (Fig. 3). 5. Wash the surface with a 50-µL injection of 1 M NaCl in filtered (0.2 µm) distilled water. 6. Allow the surface to equilibrate in HBS buffer to a stable baseline, the difference in RU between the beginning of the injection phase and the end of the wash period reflects the amount of DNA bound. For stoichiometry, and availability of sites, see Note 6. 7. Note that values in excess of 100 RU for DNA molecules of appro 100–1000 bp are to be avoided for a number of reasons (see Notes 2–6).

3.2. Protein Binding to the Immobilized DNA 1. The protein should be prepared in the required buffer over a range of concentrations, at least two orders of magnitude on either side of the suspected Kd. The detergent P20 should be present at concentrations of around 0.005% unless it has been shown to have a deleterious effect on the interaction with the DNA. 2. Samples should be injected over the both the immobilized DNA surface and a surface that has been treated with streptavidin and ethanolamine but no DNA as a blank (Fig. 4). 3. The baseline should be stable with a slope inferior to 10 RU/min. If this is not the case, then check the temperature of both the apparatus and the continuous-flow buffer. If the problem persists, replace the sensor surface with an old used chip and carry out a desorb and sanitize. Re-equilibrate the immobilized DNA chip in new filtered (0.22-µm filters) buffer by running the prime command. If the problem persists, a potential reason may be degradation of the integrated fluid cartridge necessitating its replacement (see Note 8). 4. A typical sensorgram is shown in Fig. 4. Note that at low protein concentrations it is very difficult to obtain steady-state saturation levels. Note also that there is a nonspecific interaction with the control surface (curve b) and also a contribution from the bulk refractive index effect and that both of these must be taken into consideration when deducing kinetic or equilibrium values.

4. Notes 1. The relationship between mass and refractive index changes as measured by changes in the angle at which resonance occurs in this system, although theoretically available through the additive properties of molar refractivity, has been empirically established such that 10–1° is equivalent to 1000 resonance units (RU)

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2.

3.

4.

5.

Buckle and relates to a change in mass of 1 ng of a globular protein at the surface. Using these values, a similar relationship has been demonstrated where 0.78 ng of a nucleic acid gives the same response (1000 RU) (4,5). Mass transport: In instances where the association rate is particularly elevated and the diffusion rate of the nonimmobilized molecules is not especially fast, then the interaction of the free molecules with the immobilized ligand may deplete a layer of solvent immediately surrounding the immobilized ligand such that the rate-limiting step for association now becomes the rate of repletion of this layer from the bulk solvent. Two practical solutions to this problem are first to use a low immobilization density and, second, to use relatively elevated flow rates (>20 µL/min). Estimation of the amplitude of the signal allowing calculation of the final equilibrium or steady-state level is often hampered by bulk refractive index changes that mask the initial and final phases of the injection period. Blank runs over free surfaces may be used to correct for this provided that the free surface reflects as close as possible the ligand surface. For example, if streptavidin is being used to immobilize the DNA, then a surface containing a comparable quantity of streptavidin to the sample surface should be used as a blank (Fig. 4). Incidentally, this is again an argument in favor of low levels of DNA immobilization so that the control surface is very similar in refractive properties to the surface under study. Alternatively, and indeed if possible, preferably samples being injected across the surface should be desalted into the injection buffer so as to minimize bulk refractive index effects. An ideal method is to use fast desalting columns on HPLC/FPLC systems such as the SMART (Amersham Pharmacia Biotech) which produce little dilution, are rapid and allow automatic quantification of the protein. The asymptote of the binding curve should provide the stoichiometry of the reaction assuming that all the DNA molecules are available for binding. If this is uncertain, one way of ensuring that a double-stranded target DNA is accessible would be to hybridize the protein binding site in situ on the surface by immobilizing a single strand and then hybridizing a second homologous strand to constitute the double stranded site. The mass increase in the second hybridization step would give the number of accessible DNA molecules because it results from a successful hybridization at the surface. It goes without saying however that this requires great care in eliminating nonspecific adsorption of DNA onto the surface. Steric hindrance: Let us imagine for the sake of argument that we have immobilized 1000 RU of a 100-bp double-stranded DNA molecule via biotin/streptavidin to a surface. This constitutes 1.2 × 10–14 mole of DNA or 2 × 108 molecules. Let us further assume that these molecules are evenly and randomly distributed across the 1-mm2 surface so a simple calculation shows that each molecule is separated from its neighbor by 70 nm. The DNA molecules are around 30 nm long, so if the dextran is itself flexible, then these DNA molecules are going to be in contact. This may produce problems such as occlusion of sites, creation of new potential binding sites, or unusual DNA structures possessing aberrant binding modes for


6.

7. 8.

9.

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target proteins. Even if a relatively large molecule such as a prokaryotic RNA polymerase (500 kDa, approximate diameter-10 nm) were to bind to a single site on the DNA, then there would be every possibility that adjacent bound molecules are going to interact with complicated consequences upon binding kinetics. The amplitude of each signal should be estimated manually from the steady-state signal at the end of the injection period or, if this is difficult because of a slow overall relaxation time or because of a perturbing bulk refractive index effect, from the projected Req value calculated from direct fits. Note that the projected Req values should be identical for fits of both the association and dissociation phases. It is essential that in the equilibrium analysis of these curves, the stoichiometry reflects the biology of the situation. An immobilized DNA ligand containing a single site for a protein should bind only one protein per site at saturation. If this is not the case, then no single- or double-binding site analysis is going to provide meaningful affinity values. It will be clear from careful inspection of the dissociation phase, for example, that several interactions are involved and a simple analysis cannot be made. This procedure will activate surface 1. Other surfaces can be activated simply by changing the designated flow cell. The Biacore machines are robust but susceptible to poorly prepared solutions. All solutions entering the fluid system must be filtered through 0.2-µm membranes and preferentially be sterile. The machine should be cleaned periodically with the desorb and sanitize protocols, and when not in use, it is recommended to maintain a continuous flow of distilled water containing 0.005% P20 at 20°C. As pointed out in ref. 6, there are at least three consistency tests that should be applied to a given analysis: First, the equilibrium or steady-state analysis provides a dissociation constant Kd from the Langmuir isotherm derived from Eq. 6: [P] Req [P] = R0 + (Rsat – R0) (12) Kd + [P]

where Rsat corresponds to the asymptote of binding curves of the type shown in Fig. 4. The value obtained for Kd here should be equal to that obtained from the thermodynamic relationship described in Eq. 11. In cases where this is not the case neither treatment may reflect the correct situation. Second, the use of Eq. 10 in a linear least squares analysis provides a value for koff. This value should agree with that obtained from direct analysis of the dissociation phase of all sensorgrams at all concentrations of P (Eq. 9). Third, it should be obvious from Eq. 10 that the value for koff should be inferior to that of kobs over all values of [P] because koff must always be in excess of zero. Any values that do not comply to these simple tests are thus derived from an erroneous treatment of the interaction. 10. Recapture: At high levels of immobilization, when a captured ligand dissociates from the immobilized surface, it may subsequently be recruited to an adjacent molecule. The effect of this will be to decrease the numerical values ascribed to derived dissociation constants. Thus, in practice, the density of immobilization

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Buckle must be adjusted so that the dissociation rate is independent of immobilized ligand density. If this proves difficult, then one can, with considerable error, extrapolate to infinite dilution. Finally, a free ligand may be included during the dissociation phase in order to calculate an affinity for the competitor and thus allow an estimation of a true dissociation constant.

References 1. Buc, H. (1998) L’utilisation de la résonance de plasmon de surface pour la détermination des constantes d’equilibre). Regard Biochim. 2, 21–26. 2. Schuck, P. (1997) Reliable determination of binding affinity and kinetics using surface plasmon resonance biosensors. Curr. Opin. Biotechnol. 8(4), 498–502. 3. Schuck, P. and Minton, A. P. (1996) Analysis of mass transport-limited binding kinetics in evanescent wave biosensors. Analy. Biochem. 240(2), 262–272. 4. Buckle, M., et al. (1996) Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance. Proc. Natl. Acad. Sci. USA 93(2), 889–894. 5. Fisher, R. J., et al. (1994) Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms. Protein Sci. 3(2), 257–266. 6. Schuck, P. and Minton, A. P. (1996) Kinetic analysis of biosensor data: elementary tests for self-consistency [see comments]. Trends Biochem. Sci. 21(12), 458–460. 7. Adelman, K., et al. (1998) Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle. Proc. Natl. Acad. Sci. USA 95, 15,247–15,252.

Reconstitution of Complexes

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37 Reconstitution of Protein–DNA Complexes for Crystallization Rachel M. Conlin and Raymond S. Brown 1. Introduction An increasing number of structural studies are aimed at identifying the principles that govern protein-DNA recognition in gene regulation (1). This work depends on the successful reconstitution of protein–DNA complexes from their purified components. X-ray crystallography and two-dimensional nuclear magnetic resonance (NMR) techniques require large amounts of pure protein and DNA. These can be supplied through expression in bacteria of the cDNA coding for intact proteins or their smaller DNA-binding domains and the automated chemical synthesis of DNA in the laboratory. Expression of the protein of interest is usually achieved at high levels in bacteria. An increasingly popular system is the combination of Escherichia coli strain BL21(DE3) transformed with a pRSET expression vector containing a strong phage promoter adjacent to the cloning site (2). This particular bacterial strain has an integrated T7 RNA polymerase gene that can be induced with IPTG (3). Target DNA sequences are easily identified by DNase I footprinting of a radioactively labeled DNA restriction fragment to which the protein is bound (4). Little technical difficulty is experienced in the chemical synthesis of these DNA sequences, up to about 40 base pairs in length, in amounts necessary to perform structural studies. Considerable progress has been made in solving three-dimensional structures of protein–DNA complexes (1), largely because proteins and their isolated DNA-binding domains are able to recognize and form stable complexes with quite short duplexes containing the binding sequence. Indeed, protein– DNA complexes have been crystallized that contain duplexes that are as short as 8 base pairs in length (5). From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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In this chapter, we describe the preparation of a protein–DNA complex composed of a six-zinc-finger fragment (22 kDa) of Xenopus laevis transcription factor TFIIIA and a synthetic DNA duplex. The experimental strategy, reconstitution conditions, and technical problems to be discussed are probably quite similar to those encountered with most other protein–DNA complexes. In this example, the solution properties of the protein are known and the limits of the target DNA sequence are precisely defined. It has generally been assumed that association of the components takes place spontaneously to produce protein– DNA complexes of the desired molar composition. We have chosen to perform biochemical analysis in order to optimize authentic binding and ensure the efficient scaling up of reconstitution conditions. 2. Materials 1. Expression plasmid pRSET B (Invitrogen, Carlsbad, CA). 2. Buffer A: 500 mM NaCl, 2 mM benzamidine–HCl, 1 mM dithiothreitol (DTT), 1 mM NaN3, 50% (v/v) glycerol, and 50 mM Tris-HCl, pH 7.5. 3. Standard protein solution: 1 mg/mL bovine serum albumin (BSA). 4. Protein assay concentrate (Bio-Rad, Richmond, CA). Dilute 1:5 with water and filter through Whatman 2v paper. 5. 0.01% (w/v) Coomassie brilliant blue R250 in 20% (v/v) ethanol and 10% (v/v) acetic acid. 6. 1 M Tris-HCl, pH 8.0. 7. DEAE–Sephacel (Pharmacia, Piscataway, NJ). 8. 2 M sodium acetate. 9. Repelcote solution (Hopkin and Williams, Chadwell Heath, Essex, UK). 10. Millex HA and Millex HV4 (0.45 µm) filter units (Millipore, Bedford, MA). 11. Buffer B: 100 mM NaCl and 10 mM Tris-HCl, pH 8.0. 12. Amberlite MB-150 resin (Sigma, St. Louis, MO). 13. 150-mL (0.2-µm) NYL/50 filter unit (Sybron Corp., Rochester, NY). 14. Thermal cycler for polymerase chain reaction (PCR). 15. 5X binding buffer: 250 mM NaCl, 5 µM MgCl2, 5 mM DTT, 50 µM zinc acetate, 50% (v/v) glycerol and 100 mM Tris-HCl, pH 7.5. 16. 1 M HEPES–NaOH, pH 7.5. 17. 5 M NaCl. 18. Buffer C: 100 mM NaCl, 1 mM DTT, 1 mM NaN3, and 20 mM Tris-HCl, pH 7.5. 19. Collodion bags and 300-mL glass vacuum dialysis flask (Sartorius AG, Göttingen, Germany). 20. Magnetic micro flea 5-mm × 2-mm spinbar (Bel-Art products, Pequannock, NJ). 21. Microcon-10 microconcentrators (Amicon, Beverly, MA). 22. Natrix nucleic acid sparse matrix kit (Hampton Research, Lagnua Niguel, CA). 23. Linbro tissue culture multiwell plate with cover. Twenty-four flat-bottomed wells (1.7 cm × 1.6 cm) (Flow Laboratories, McLean, VA). 24. AquaSil water-soluble siliconizing fluid (Pierce, Rockford, IL).


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25. DiSPo plastic cover slips M6100 (American Scientific Products, McGaw Park, IL). 26. High-vacuum grease (Dow Corning, Midland, MI). 27. A 10cc plastic B-D syringe (Becton-Dickinson, Rutherford, NJ).

3. Methods 3.1. Purification of a Recombinant DNA-Binding Protein Standard laboratory techniques for protein purification (6) will not be described in detail in this chapter. Advantage is usually taken of the rather basic nature of DNA-binding proteins to isolate them from the bacterial cell extract. Few of the bacterial proteins bind so strongly to chromatography matrices, such as CM–Sepharose, Bio-Rex 70, S-Sepharose, phosphocellulose, and hydroxyapatite–Ultrogel. The protein of interest can usually be eluted with a NaCl gradient. Affinity chromatography with heparin–Sepharose, an immobilized dye–Sepharose, DNA–agarose or phenyl–Sepharose often provides an adequate final purification step.

3.1.1. Preparation of Protein The cDNA for the 22-kDa fragment of TFIIIA (amino acids 1–190) was cloned into the vector pRSET B and expressed in strain BL21 (DE3) (see Note 1). After sonication, the cell pellet is stirred in 0.5 M NaCl and 7 M urea for 48 h at 4°C. Purification of the protein (Fig. 1) is carried out in 7 M urea on columns of Bio-Rex 70 and heparin–Sepharose (see Notes 2–4). Workup of the protein for use in DNA binding is described in detail as follows: 1. Pool those fractions that contain protein after heparin–Sepharose column chromatography. 2. Concentrate the protein to 5 mg/mL by vacuum dialysis at 4°C in a collodion bag (see Note 5). 3. Dialyze against ice-cold buffer A. 4. Store the protein at –20°C in a 1.5-mL Eppendorf tube.

3.1.2. Measurement of Protein Concentration 1. Construct a calibration curve of protein concentration versus absorbance from samples (in triplicate) containing 15, 25, 35, and 45 µL of the standard protein solution and water added to 100 µL in glass tubes (see Note 6). 2. Add 5 mL of protein assay reagent and read the absorbance at 595 nm with a spectrophotometer after the blue color has developed for 10 min. 3. Calculate the relative concentration from comparison of the values for the protein sample with the calibration curve (see Notes 7 and 8). 4. Examine the purity of the protein sample by standard sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. A 5% stacking/13.5% resolving slab gel (30:0.8 acrylamide:bis) is suitable for this purpose (see Note 9). At least 0.1 µg

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Fig. 1. (A) 13.5% SDS-PAGE gel showing the expression of TFIIIA (residues 1–190) in E. coli BL21(DE3). Dalton markers are in lane 1. Whole-cell extracts are run in lanes 2 and 3 before and after induction with 1 mM IPTG. The TFIIIA protein after purification is shown in lane 4. (B) 6.5% PAGE mobility shift gel for analysis of protein-DNA binding. One microgram of the synthetic 31-mer DNA duplex was mixed with 0.5 µg (lane 1) or 1 µg (lane 2) of the 22.1-kDa TFIIIA fragment. Bands that contain DNA are visualized by ethidium bromide stain and UV illumination. of protein is detected in a band with Coomassie blue stain. If necessary, adjust the protein concentration by an estimate of its gel purity.

3.2. Preparation of Synthetic Oligomers Typically a 1-µmol scale synthesis with a commercially available machine provides 1–2 mg of a purified 31-mer. The standard laboratory methods for isolation of the 5'-dimethoxytrityl full-length oligomer and subsequent chemical removal of base protecting groups will not be described here (see Note 10). Oligomers pure enough for this work can be obtained with a fast protein liquid chromatography system (FPLC) and suitable columns (7).

3.2.1. Recovery and Concentration of Oligomers 1. Adjust the pH to 8.0 and apply the pooled Mono Q column fractions containing the oligomer onto a 0.5-mL DEAE-Sephacel column equilibrated with 50 mM Tris-HCl (pH 8.0) at room temperature.


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2. Elute the bound oligomer with 5X 1 mL of 2 M sodium acetate into a silanized 30-mL Corex tube. 3. Add 20 mL of 95% ethanol and precipitate the oligomer overnight at –20°C. 4. Centrifuge (45 min at 7500g, 4°C) to recover the oligomer. 5. Wash the pellet with 25 mL of 75% ethanol to remove excess sodium acetate and centrifuge as in step 4. Pour off the supernatant and dry pellet under vacuum. 6. Redissolve the oligomer in 1 mL of water (see Note 11) and then filter through a Millex HV4 unit (0.45 µm). 7. Measure the absorbance at 260 nm with a spectrophotometer (dilute the oligomer as necessary with 100 mM NaCl). The concentration is calculated with a conversion factor of 25 A260 units = 1 mg. Store the oligomer at 1 mg/mL at –20°C.

3.2.2. Annealing the Duplex 1. Mix equal amounts of complementary DNA strands in 100 µL of buffer B at 1 mg/mL in 0.25-mL PCR tubes. 2. Heat at 95°C for 5 min and then slowly cool to 4°C (see Note 12). 3. Examine duplex formation by electrophoresis at 50 V in a nondenaturing 6.5% polyacrylamide slab gel at room temperature. One-tenth microgram of DNA in a gel band is easily visible after staining in ethidium bromide (1 mg/L) and illumination with ultraviolet (UV) light.

3.3. Testing the Reconstitution Conditions The duplex is titrated with increasing amounts of protein in order to measure DNA-binding activity (Fig. 1). The resulting complexes are monitored by mobility shift on a nondenaturing 6.5% polyacrylamide gel (see Notes 13–15). This method is used to systematically optimize 1:1 molar complex formation with respect to incubation time, temperature, concentration of monovalent and divalent ions, pH, and the presence of glycerol and nonionic detergents like Nonidet P-40. 1. Mix 1 µg of DNA, 2 µL of 5X binding buffer, and 5 M NaCl (to make 0.5 M NaCl after step 2) in a 1.5-mL Eppendorf tube. 2. Add 0.5, 1, 1.5, and 2X molar excess of protein and stir in gently with the Pipetman tip (see Note 16). 3. Dilute with 10 µL of 1X binding buffer and incubate at room temperature for 15 min. 4. Apply the 20-µL samples, without tracking dyes, to a nondenaturing 6.5% polyacrylamide gel (see Note 17). 5. Load dyes (bromophenol blue and xylene cyanol FF) in adjacent tracks to indicate progress of the electrophoresis. The protein–DNA complex migrates between the dyes. 6. Stain the gel first with ethidium bromide (1 mg/L) for DNA and then with 0.01% Coomassie blue for protein.

3.3.1. Scaling-Up Reconstitution of the Complex In low-salt conditions, the protein shows a strong tendency to aggregate. This is detected as material trapped at the top of the nondenaturing polyacryla-

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mide gel. An excess of protein results in formation of some protein–DNA complexes with higher molar stoichiometries. We have discovered that these unwanted effects can be substantially eliminated by dilution of NaCl in the scaled-up reconstitution mix in steps; from 0.75 M to 0.225 M NaCl (see Note 18). 1. Mix together 500 µg of DNA, 200 µL of 5X binding buffer, and 5 M NaCl water in a 50-mL polypropylene Falcon tube (the final reconstitution mix, including the protein, is 0.75 M NaCl). 2. Add 625 µg of protein and mix together by gentle swirling. 3. Dilute with 1 mL of 1X binding buffer and incubate for 5 min at room temperature. 4. Add 1 mL of 1X binding buffer and incubate for 5 min. 5. Add 1 mL of 1X binding buffer and incubate for 5 min. 6. Concentrate the complex to 10 mg/mL by centrifugation 4°C with Microcon-10 microconcentrator units (Amicon).

3.3.2. Crystallization Trials Crystallization conditions are often screened according to an incomplete factorial design (8). Supplies and kits are available from Hampton Research. Some typical crystallization results, mainly employing the hanging drop/vapor diffusion method, are shown in Table 1. Conditions are screened at 4°C and at room temperature with small droplets (1–2 µL) of the protein–DNA complex in which the concentrations of salts, buffers, and precipitants are systematically varied by small amounts. Crystallization often occurs at or close to the point of precipitation of the protein–DNA complex. 1. Pass all solutions of salts, buffers and precipitants through 0.45 µ filters (Millex HA units) before use. 2. Blow dust from plastic pipet tips and then place 1-µL aliquots of the protein– DNA complex onto silanized plastic cover slips (see Note 19). 3. Mix with 1 µL of appropriate salts, buffer and precipitant or the well solution (see Note 20). 4. Apply vacuum grease to the upper rim of the wells of the tissue culture plate (see Note 21). 5. Into each well put 1 mL of suitable precipitant. 6. Carefully invert a cover slip without disturbing the droplet and place onto the greased rim so as to seal each well. 7. Store the tissue culture plates in closed polystyrene boxes to avoid excessive vibration and changes in temperature. 8. After a week inspect the droplets for signs of crystallization with a stereo microscope at ×50 magnification.

4. Notes 1. Bacteria grown in LB broth are induced by addition of 1 mM IPTG and 100 µM zinc acetate for 2 h at 37°C. 2. Losses of protein during column chromatography may be decreased by addition of 10% (v/v) glycerol.


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Table 1 Crystallization Conditions for Protein–DNA Complexes Protein–DNA Complex

Method

Sso7d: 8-mer, Gao et al. (5) NF-κB p50/p65: 12-mer, Chen et al. (9)

DNA and protein in 2 mM Tris-HCl, pH 6.5, and 2.6% PEG 400 at room temperature Final conditions are 50 mM sodium acetate (pH 5.5) 100 mM CaCl2, 0.125% β-octyl-glucoside, 1 mM spermine, 10 mM DTT, and 8% PEG 3350 at 18°C Mix protein and DNA in 5 mM bis-tris– propane (pH 7.0) with 1 vol of well solution at room temperature

Antp homeodomain: 15-mer, Fraenkel and Pabo (10) Stat3beta: 18-mer glycerol, Becker et al. (11)

DNA and protein in 20 mM HEPES (pH 7.0) 200 mM NaCl, 10 mM MgCl2, 5 mM DTT, and 0.5 mM PMSF. Mix with 1 volume of well solution at room temperature

NFAT/AP–1: 20-mer, Chen et al. (12)

Final conditions are 10 mM HEPES (pH 7.5), 1 mM DTT, 100 mM NaCl, 20% (v/v) glycerol, 500 mM ammonium acetate Mix protein and DNA in 1 mM EDTA, 1 mM DTT, 20 mM Tris-HCl, pH 8.0, and 0.001% NaN3 with 1 vol of the well solution at 20°C

GABPalpha/beta: 21-mer, Batchelor et al. (13)

Topo I: 22-mer Redinbo et al. (14)

T7 DNA polymerase: 21-mer + 26-mer Doublie et al. (15)

TFIIIA (residues 1–190): 31-mer, Nolte et al. (16)

DtxR repressor: 33-mer, White et al. (17)

Mix 1µL of DNA in 6 mM NaCl with 2 µL of protein in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM DTT, and 3 µL H2O. Add to 3 µL of well solution at 22°C Microseeding

Final conditions are 165 mM NaCl, 35 mM sodium acetate, 3.2 mM DTT, 9.2% (v/v) glycerol, 1.8 mM NaN3, 1.8 mM cadaverine–2HCl, 5.5 mM Tris-HCl (pH 8.0), and 22.5% PEG 4000 at 18°C Mix 2µL of protein and DNA in 1 mM NiCl2, and 100 mM Tris-HCl, pH 7.5 with 1 µL well solution at room temperature

Well solution 15% PEG 400

1–5% MPD 20 mM bis-tris– propane (pH 7.0), and10 mM NiCl2 10% v/v glycerol, 0.1–0.4 M NaCl. 5 mM MgSO4, 50 mM MES, pH 5.6–6.0, 0.1 M ammonium aceta te 300 mM ammonium acetate 100 mM bis-tris– propane, pH9.0, 5 mM cobaltic hexammine chloride, and 9% PEG 1000 100 mM MgCl2, 10 mM DTT, 100 mM Tris-HCl (pH 7.7), and 24% PEG 400 100 mM ACES, pH 7.5, 30 mM MgCl2, 120 mM ammonium sulfate, 5 mM DTT, and 12% PEG 8000

6–12% PEG 4000, 10 mM MgCl2, and 100 mM MES (pH 6.0)

Abbreviations: ACES: 2-[(2-Amino–2-oxoethly)amino]ethanesulfonic acid; DTT: dithiothreitol; EDTA: disodium ethylenediaminetetraacetic acid; HEPES: (N-[2-hydroxyethyl]piperazine-N'[2-ethanesulfonic acid]); MES: 2-[N-morpholino]ethane-sulfonic acid; MPD: 2-methyl-2, 4-pentanediol; PEG: polyethylene glycol; PMSF: phenylmethylsulfonyl flluoride.

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3. Customized gradients can be supplied to standard laboratory chromatography columns using a programmed FPLC control unit and pumps. Flow rates of 1–50 mL/h are possible without significant back pressure. 4. The protein binds to heparin–Sepharose in 0.25 M NaCl and 7 M urea. A reverse gradient is applied to remove urea. The protein is eluted in a 0.25 M NaCl to 2 M NaCl gradient and 10 µM zinc acetate. 5. The collodion bag is pre-equilibrated in buffer and contains a small magnetic stirrer (5 mm × 2 mm) to aid dialysis and recovery of the protein. 6. This assay can be used to measure protein concentrations between 50 µg/mL and 50 mg/mL according to the manufacturer’s directions. 7. Data may be conveniently analyzed by linear regression using commercial software like KaleidaGraph. 8. The protein concentration obtained by the Bradford colorimetric assay is relative. An absolute value requires multiplication by a conversion factor. This factor can be derived from simultaneous amino acid analysis or microKjeldahl nitrogen determination (18). In the case of the TFIIIA 22-kDa fragment, the deduced concentration of BSA is multiplied by 0.39. 9. TFIIIA and its fragments have anomalous electrophoretic mobilities. Reduction and carboxymethylation with 50 mM iodoacetamide restores the predicted gel mobility to the intact protein. Fragments containing zinc fingers generally run slower than expected after this treatment. 10. Chemical detritylation can be efficiently performed by passing 0.5% trifluoroacetic acid for 3 min at room temperature through a reverse-phase ProRPC HR column (Pharmacia). Following deprotection of the bases (30% [w/v] NH4OH, 16 h at 65°C), the oligomer is purified with a Mono Q HR column (Pharmacia) and eluted in a NaCl gradient with 7 M urea according to the manufacturer’s instructions. 11. After the addition of water, the Corex tube is sealed with parafilm to avoid spillage. The water is rolled around the walls of the silanized tube as well as on the pellet. 12. This is conveniently done in a thermal cycler PCR machine at a linear cooling rate of 1°C/3 min. 13. A stock acrylamide solution (30:0.8 acrylamide:bis) is deionized with Amberlite MB–150 resin (10 g/100 mL) for 1 h, filtered through a NYL/50 filter unit (0.2 µm), and stored at 4°C. This treatment minimizes the sequestering of the protein from protein–DNA complexes during gel electrophoresis. 14. Glass plates, combs, spacers and the gel apparatus must be cleaned and washed completely free of detergent to avoid disruption of the protein–DNA complex. 15. The polyacrylamide gel is 1.5 mm thick and contains 50 mM NaCl, 40 mM HEPES–NaOH (pH 7.5), and 5% (v/v) glycerol. The running buffer consists of 50 mM NaCl, 20 mM HEPES–NaOH (pH 7.5). 16. A duplex with an unrelated sequence of the same length may be used to monitor the level of nonspecific protein binding. The affinity of the protein for each of the single strands of the duplex can also be tested. 17. Samples can be applied smoothly to the gel by slowly winding the Pipetman volume control back to zero.


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18. At high salt concentration, above 0.65 M NaCl, the protein–DNA complex is dissociated and both components are soluble. The salt concentration is lowered stepwise to 0.225 M NaCl to reconstitute the complex (19). At the same time, the protein is diluted and becomes less likely to aggregate or bind nonspecifically to the duplex. 19. Wash dust off the cover slips with deionized water and dry in a suitable rack at 50°C in an oven. Immerse cover slips for 15 min in dilute AquaSil (1:40) and leave to dry overnight. Wash in deionized water and dry at 50°C. Keep in a closed container to avoid contact with dust. 20. It is customary to equilibrate against a well solution that contains double the concentration of the droplet components. One volume of the well solution containing appropriate salts, buffers, and precipitant is added to the droplet of the protein–DNA complex. 21. The grease can be applied accurately to the rim with a 10cc plastic syringe filled with high-vacuum grease.

References 1. Steitz, T. A. (1990) Structural studies of protein-nucleic acid interaction: the sources of sequence-specific binding. Quart. Rev. Biophys. 23, 205–280. 2. Dubendorff, J. W., and Studier, F. W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59. 3. Studier, F. W., Rosenberg, A. G., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct the expression of cloned genes. Methods Enzymol. 85, 60–89. 4. Engelke, D. R., Ng, S.-Y., Shastry, B. S., and Roeder, R. G. (1980) Specific interaction of a purified transcription factor with an internal control regionof 5S RNA genes. Cell 19, 717–728. 5. Gao, Y. G., Su, S. Y., Robinson, H., Padmanabhan, S., Lim, L., MacCrary, B. S., et al. (1998) The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA. Nature Struct. Biol. 5, 782–786. 6. Deutscher, M. P. (ed.) (1990) Guide to Protein Purification, Methods in Enzymology, Academic, New York, p. 182. 7. Oliver, R. W. A. (ed.) (1989) HPLC of macromolecules: a practical approach. IRL, Oxford. 8. Carter, C. W.,Jr. (1992) Crystallization of Nucleic Acids and Proteins: A Practical Approach (Ducruix, A. and Giege, R., eds.), IRL, NY, pp. 47–71. 9. Chen, F. E., Huang, D.-B., Chen, Y.-Q., and Ghosh, G. (1998) Crystal structure of p50/p65 heterodimer. Nature 391, 410–413. 10. Fraenkel, E. and Pabo, C. O. (1998) Comparison of X-ray and NMR structure for the Antennapedia homodomain–DNA complex. Nature Struct. Biol. 5, 692–696. 11. Becker, S., Groner, B., and Muller, C. W. (1998) Three-dimensional structure of the Stat3beta homodimer bound to DNA. Nature 394, 145–151.

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12. Chen, L., Glover, J. N. M., Hogan, P. G., Rao, A., and Harrison, S. C. (1998) Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392, 42–48. 13. Batchelor, A. H., Piper, D. E., de la Brousse, F. C., McKnight, S. L., and Wolberger, C. (1998) The structure of GABPalpha/beta: an ETS domain-ankyrin repeat heterodimer bound to DNA. Science 279, 1037–1041. 14. Redinbo, M. R., Stewart, L., Kuhn, P., Champoux, J. J., and Hol, W. G. J. (1998) Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279, 1504–1513. 15. Doublie, S., Tabor, S., Long, A. M., Richardson, C. C., and Ellenberger, T. (1998) Crystal structure of a bacteriophage T7 DNA replication complex at 2. 2Å resolution. Nature 391, 251–258. 16. Nolte, R. T, Conlin, R. M., Harrison, S. C., and Brown, R. S. (1998) Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex. Proc. Natl. Acad. Sci. USA 95, 2938–2943. 17. White, A., Ding, X., vanderSpek, J. C., Murphy, J. R., and Ringe, D. (1998) Structure of the metal-ion-activated diphtheria toxin repressor/tox operator complex. Nature 394, 502–506. 18. Jaenicke, L. (1974) A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal. Biochem. 61, 623–627. 19. Zwieb, C. and Brown, R. S. (1990) Absence of substantial bending in Xenopus laevis transcription factor IIIA-DNA complexes. Nucleic Acids Res. 18, 583–587.

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38 Two-Dimensional Crystallization of Soluble Protein Complexes Patrick Schultz, Nicolas Bischler, and Luc Lebeau 1. Introduction Structural data on biological macromolecules provide invaluable insights into the interactions of proteins with nucleic acids. Data obtained at atomic resolution by X-ray diffraction or nuclear magnetic resonance (NMR) studies ultimately describes the exact folding of polypeptide chains and the contacts between proteins and DNA. However, many complexes are difficult to analyze at atomic resolution because either they are too large or too difficult to crystallize in three dimensions (3-D). Recent progress in electron microscopy, specimen preservation, and image processing provides the possibility to calculate detailed molecular envelopes that are complementary to X-ray crystallography with little theoretical limit on specimen size. In the case of membrane proteins organized into one-molecule-thick two-dimensional (2-D) arrays, atomic models could be elaborated from electron microscopy data (1,2). It is beyond the scope of this chapter to describe in detail the electron crystallographic methods and computer image analysis required to calculate a 3-D model of a crystallized protein complex; these aspects are described elsewhere (3). Here, we will focus on the formation of 2-D crystals of soluble proteins, an essential preliminary step in high-resolution structure determination by electron microscopy, and provide the necessary information to set up, screen, and evaluate crystallization experiments. The remarkable achievements in the field of membrane protein 2-D crystals prompted the pioneering work of Kornberg and collaborators aimed at transposing the crystallization mechanisms occurring in a lipid bilayer to soluble proteins (4). The method consists in targeting the protein of interest to a lipid surface self-assembled as a monomolecular film at an air–water interface. From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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The buffer-exposed hydrophilic part of the lipid molecule may carry charged groups (5,6) or be chemically modified (7,8) in order to interact with the protein. Consequently, the protein is concentrated at the lipid plane and adopts only a few orientations relatively to it. If the lipid molecules are free to diffuse in the monolayer plane, the system permits the 2-D crystallization of the macromolecule. This achieved, the lipid–protein film is transferred onto a solid support and processed for electron microscopy observations. As was demonstrated by the study of streptavidin 2-D crystals (9), which revealed structural information down to 3 Å in projection, such an approach can yield high-resolution structural data. Two categories of lipid derivatives can be used according to the specificity of the interaction to be established with the protein complex (10). On the one hand, a charged surface can be created by using lipids with positive or negative charges that will interact with the surface potential of the protein to be crystallized (5). The characteristic behavior of the protein in ion-exchange chromatography may give a hint as to which type of charged lipids should be used. Typically used lipids are phosphatidyl serine which carries a negative charge, and stearyl amine or alkyl trimethyl ammonium, both of which are positively charged. On the other hand the lipid film may be derivatized by a specific ligand recognized by the protein of interest. The ligand is chemically grafted to the lipid moiety through a linker whose length modulates the accessibility for the protein. The grafted molecule can be a natural ligand of the protein such as dinitrophenol for specific anti-DNP antibodies (4), novobiocin for gyrase B subunit (8) (Fig. 1), or biotin for streptavidin (11). More recently lipid molecules have been designed to interact with specific tags (such as polyhistidines) introduced genetically into the sequence of the protein of interest (12,13). The polar head group of the lipid molecule carries a nitrilotriacetate moiety, which chelates nickel ions and interacts with the histidine tag. Stability and fluidity of the spread lipid layer are mainly provided by the hydrophobic part of the lipid (Fig. 1). The lipid layer has to be in the fluid phase at the incubation temperature because crystallization of the lipid chains was shown to prevent protein organization probably by lowering its 2-D mobility (11,13). In most cases, a cis double bond in the alkyl chains provides sufficient fluidity. In addition, the lateral cohesion of the lipid molecules has to be strong enough to allow the spreading of a stable monolayer and prevent the solubilization of the lipid as micelles or liposomes. A double alkyl chain containing 18 carbon atoms (dioleyl) generally fulfills these requirements. When protein–lipid interactions occur, the proteins are rapidly concentrated close to the lipid layer and are likely to be partly oriented. In the case of a specific interaction with a functionalized lipid, the macromolecules are tethered to the lipid film by a unique site and the extent of orientation is deter-


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Fig. 1. Schematic representation of a lipid molecule used for 2-D crystallization of the b-subunit of DNA gyrase. The hydrophobic alkyl chain with a cis double bond confers fluidity and stability to the spread lipid layer. The linker region provides accessibility of the ligand for the protein of interest. The recognition function, here a novobiocin molecule, determines the interaction properties between the lipid and the protein.

mined by the length and flexibility of the linker region (8) (Fig. 1). Increased concentration, possible preferential orientation, and in-plane mobility facilitate contacts between macromolecules, which results in their increased organi-

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zation when an ordered network of interactions is established. However, a dead end may be reached if the macromolecules interact too strongly or too rapidly with each other. As a consequence, 2-D aggregates can form, which appear as close-packed, noncrystalline assemblies. The method requires limited amounts of protein because a single experiment may only need 300 ng of protein. However, a quantity of 1 mg is more realistic because any new project implies systematic trials starting without a priori knowledge of the factors affecting the lipid–protein interactions. When a specific interaction of the protein with the lipid is involved, the degree of purity of the biological sample appears to be of less crucial importance than when charged lipid are used (13,14). 2. Materials 1. A suitable lipid to interact with the protein of interest. 2. Teflon® or Nylon® blocks in which cylindrical wells 4 mm in diameter and 1 mm deep have been milled such that each well can contain about 15 µL of aqueous solution (Fig. 2). 3. Standard Cu or Cu/Rh 300 mesh electron microscopy grids. 4. Mica sheets 2.5 × 9 cm in size. 5. A carbon evaporator. 6. A 2% uranyl acetate solution. 7. A control electron microscope. 8. An optical diffraction bench.

3. Methods 3.1. Preparation of Electron Microscopy Supports The lipid–protein assemblies will have to be transferred onto a standard electron microscopy grid. The grids need to be coated with a thin hydrophobic carbon film to support the assemblies and to allow the adsorption of the hydrophobic lipid alkyl chains. 1. The mica sheets are freshly cleaved to create a clean and flat surface. 2. The mica sheets are placed into a carbon evaporator and a 10 to 50-nm thick carbon film is evaporated under vacuum onto the cleaved face of the sheets. 3. Electron microscopy grids are placed on a supporting filter paper below the surface of a water bath. The size of the filter paper matches that of the mica sheet and will hold a total of 75–100 grids. 4. The carbon foil is floated at the clean air–water interface by dipping the mica sheet in the water bath with an angle of about 30°. 5. Finally, the carbon foil is gently lowered onto the grids by removing the water using a vacuum pump.

It was observed with some systems, such as streptavidin, that the contact of the lipid layer with the carbon foil interferes with the quality of protein arrays


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Fig. 2. Design of a Teflon® block for 2-D crystallization experiments. (A) A Teflon® cylinder 4 cm in diameter truncated into 1 cm-thick slices and 16 wells, 4 mm in diameter and 1 mm deep, are milled into the block such that each well can contain about 15 µL of solution. (B) During the crystallization experiments performed in the wells (a), the Teflon® block (b) is placed into a humid chamber consisting of a reverted Petri dish containing some buffer (c).

(15). “Holey” carbon grids can then be used to transfer the crystals without interactions with a carbon surface, the lipid–protein layer being spread over the holes. A protocol to prepare “holey” carbon films is as follows (16): 1. The surface of an optical microscope glass slide is extensively cleaned by boiling in an aqueous detergent solution and extensive rinsing with demineralized water (H2Od). 2. The slide is immersed in a 0.1% Triton X405 (Sigma) solution for 30 min, briefly rinsed with H2Od to remove the excess of detergent and left to dry. This will result in a clean hydrophobic surface. 3. The slide is placed on a precooled aluminum block to allow minute water droplets to form on the surface by condensation. The size of the droplets depends on the humidity of the room and on the condensation time.

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4. One mL of a cellulose acetate or cellulose butyrate solution (0.4% [w/v] in ethyl acetate) is poured with a pipet over the surface, excess solution is removed from one end of the slide by touching a filter paper and left to dry. Upon drying, the cellulose forms a thin film around the water droplets, thus forming holes. 5. At this point, an optical microscope can be used to check the size of the holes and their distribution. 6. The slide is then immersed 30 min in a 0.5% (w/v) sodium dodecyl sulfosuccinate solution to peal off the cellulose film. 7. Electron microscopy grids are deposited on a supporting filter paper just below the surface of a water bath. 8. The holey cellulose film is floated on the clean air–water interface and deposited onto the grids. 9. A 50-nm-thick carbon film is evaporated onto the cellulose-coated grids. 10. The cellulose film is finally dissolved by placing the grids on a ethyl acetatesoaked filter paper.

3.2. Crystallization Experiments 1. The lipids are best stored as a dry powder under an argon atmosphere at –80°C. A mother solution at a concentration of 10 mg/mL is produced by solubilizing the lipids in an organic solvent such as a 1:1 mixture of chloroform:hexane. This solution can be stored under argon up to 1 yr at –20°C. The working lipid solution is at a concentration of 0.5–1 mg/L in an organic solvent. All solution are stored in 2 mL glass vials with Teflon® caps to prevent solvent evaporation. 2. The Teflon® wells have to be cleaned prior to use for crystallization experiments to remove residues of proteins or lipids. The Teflon® support should be dipped into a sulfochromic acid solution for 1 h, rinsed 10 times with H2 Od, dipped for 1 h into H2Od, and rinsed again three times with H2Od. Alternatively, the support can be rinsed 10 times with methanol to eliminate proteins, 10 times with a chloroform:methanol 2:1 or hexane:methanol 9:1 solution to remove lipids and rinsed 10 times with hexane to remove fatty acids. Finally, the support is brought into contact with a filter paper to remove the excess of H2Od or organic solvent, without wiping to avoid electrostatic charging, and is allowed to dry in a dustfree chamber. 3. Incubations are performed in a humid chamber to prevent buffer evaporation (Fig. 2B). The Teflon® block is placed in a reverted Petri dish containing some buffer with an opening in the top to let air in and out during removal of the lid. 4. In each well, 10 µL of buffer are added (Fig. 3B). 5. To spread the lipid at the air-buffer interface, 1 µL of the lipid solution at a concentration of 0.5–1 mg/mL is placed on the top of the drop of buffer with a micropipet. At this moment it can be observed that the surface tension of the drop is released (Fig. 3C). 6. The organic solvent is allowed to evaporate for 5 min. 7. The protein solution (5 µL) is injected into the aqueous phase (Fig. 3D). The final protein concentration is generally set between 20 and 200 µg/mL.


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Fig. 3. Setup of a crystallization experiment. In each well (A), a 10-µL drop of buffer is placed (B). Because the Teflon well is hydrophobic, the drop does not wet the surface. Upon the addition of 1 µL of the lipid solution at a concentration of 0.5–1 mg/mL, the surface tension of the drop is released (C). After evaporation of the organic solvent, 5 µL of the protein solution is injected into the well (D) and is allowed to interact with the lipid layer. The resulting lipid–protein assembly is transferred to a carboncoated electron microscopy grid placed on the top of the drop (E). 8. The incubation chamber is closed, and if oxidation is a problem air is replaced by argon. 9. The incubation time will vary from one system to another but is generally in the range of 1–36 h. Most experiments can be performed at room temperature, but longer incubation times at 4°C may improve crystal quality in some cases.

3.3. Electron Microscopy 1. The 2-D crystal is transferred to the electron microscopy grid through hydrophobic contacts between the lipid chains and the carbon foil (Fig. 3E). This is simply done by placing the grid over the well for 1–2 min. The grid is then withdrawn and prepared for observation (see Notes 1 and 2). 2. To be visualized by electron microscopy, the specimen has to be contrasted by creating a mould of heavy atoms around the proteins, a process named negative

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staining. The transferred specimen placed on the grid held by forceps is washed with a drop of buffer that is quickly removed. The buffer is then replaced by a drop of a 1–2% aqueous solution of uranyl acetate and the grid is dried by touching a piece of filter paper with the edge of the grid (see Notes 3 and 4). 3. The crystallization experiments have then to be evaluated in terms of protein concentration on the lipid layer and degree of organization (see Notes 11 and 12). When the specimen is large enough (> 50 kDa), individual molecules can be identified visually during electron microscopy inspection. To ascertain that the specimen is specifically bound to the lipid layer and not in a nonspecific way to the carbon film, it is useful to locate breaks in the lipid layer in order to observe a difference in binding efficiency with the underlying carbon film. A frequently observed intermediate step in specimen ordering is the formation of symmetryrelated oligomers that arise when a particular set of protein–protein interactions is energetically favored. The formation of oligomers probably favors further organization because interactions between symmetry related surfaces will propagate forming linear polymers or 2-D crystals. Once larger crystalline areas are obtained, electron micrographs are recorded and the extent of order is evaluated by optical diffraction.

3.4. Feedback Loops 1. If the protein is not concentrated on the lipid film, it is advisable to act on the lipid region involved in protein recognition, on its environment, or on the buffer composition. In the case of a specific lipid, the linker may be too short to allow the ligand to be recognized by the protein. Alternatively, the surface potential created by the lipid layer may have a repulsive effect on the protein and it may be of importance to modify the environment by the addition of a dilution lipid. Finally, the ionic strength of the buffer may be too high and screen electrostatic interactions between the protein and the charged lipid (see Note 5). 2. When the protein tends to form close-packed arrays, which do not evolve toward organized protein patches, it is advisable to reduce the kinetics of protein concentration either by increasing the viscosity of the medium by adding glycerol (up to 40%) or by reducing the temperature or the protein concentration. The specific or charged lipid can also be diluted with a neutral lipid to reduce the local concentration of ligand or the charge density of the surface (see Note 10). 3. The experiment has to be evaluated further in terms of macromolecular organization. Higher degrees of order are recognized visually during the electron microscopy inspection of the specimen by the appearance of patches of ordered arrays (Fig. 4A). Once larger crystalline areas are obtained, electron micrographs are recorded and the extent of order is evaluated by optical diffraction. A large number of parameters such as the pH, the ionic strength, the buffer composition, the presence of divalent cations, the protein concentration, the presence of glycerol, the incubation temperature, or the incubation time can be modified to improve crystal order (see Note 6). At this stage, the homogeneity of the specimen suspension may be crucial.


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Fig. 4. Evaluation and exploitation of a 2-D crystallization experiment. Histidinetagged yeast RNA polymerase I was incubated with nickel chelating lipids. (A) Lowmagnification electron microscopy image showing the organization of the protein complex into domains. The bar represents 5 µm. (B) A higher magnification reveals ordered RNA polymerase arrays. The bar represents 50 nm. (C) A noise-free image is obtained by averaging multiple molecular images. The stain excluding protein densities are in white and represented as lines of equal densities. (D) A 3-D model of the protein complex can be calculated by combining several views of the macromolecule obtained by tilting the crystals in the microscope. The bar represents 10 nm in (C) and (D). 4. In the case of streptavidin, the method for preparing the sample for electron microscopy and, in particular, the transfer mode proved to be essential to recover a large number of highly ordered crystals (15). More generally, the manipulation of one-molecule-thick assemblies during transfer to the electron microscope is likely to introduce at least some of the defects observed in 2-D crystals such as rotational and translational distortions, fragmentation, and other forms of disorders (see Notes 7–9). 5. An improvement of the interpretable resolution once the specimen diffracts to about 0.5 nm–1 will probably need a change in the method of specimen preservation from negatively stained to frozen hydrated samples (17).

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6. To calculate a 3-D model, astigmatism-free and well-focused images of the oriented or crystallized macromolecules must be recorded under minimal exposure conditions and generally at low temperature. These images are analyzed to calculate a noise-free image representing a projection of the macromolecular densities (Fig. 4C). Because the particles are adsorbed on a planar surface, tilted views are then recorded to recover the information normal to the lipid plane. The images are then processed and the different views are merged into a 3-D model (3) (Fig. 4D).

4. Notes 1. A good macroscopic indication that proteins bind to the lipid layers and that the transfer is efficient is obtained by visual inspection of the carbon surface after transfer. The original hydrophobic grid becomes hydrophilic, as assessed by the change in its wetting properties. 2. Storage of the carbon-coated grids in hexane atmosphere may provide higher reproducibility in the specimen transfer step by preventing adsorption of contaminating material. 3. Do not use phosphate buffers or buffers with high ionic strength, which precipitate uranyl salts. 4. Other heavy metal solutions can be used for negative staining such as sodium phosphotungstate or ammonium molybdate. 5. It is useful to check the specificity of the protein–lipid interactions. In the case of charged lipids, the protein binding should be reduced by increasing the ionic strength. In the case of functionalized lipids, the amount of transferred protein should diminish by adding some competing ligand in solution. Note that in the case of nickel-chelating lipids, it was observed that addition of small amounts of imidazole prevented the nonspecific aggregation of the protein and allowed the selection of the specific interaction with the polyhistidine tag (13). 6. Detergents should be avoided in the incubation buffer because they may solubilize the lipid layer. 7. In some cases, it was observed that the grid side on which the carbon foil was deposited affected crystal transfer (13). This effect may be related to the surface roughness of the carbon foil and of the grid (18). 8. To strengthen the crystals, 1 µL of a 0.5% glutaraldehyde solution can be added to the incubation drop before placing the electron microscopy grid in order to crosslink the specimen. 9. Another method of specimen transfer is the loop method (19). A loop is formed with a thin Pt/Pd wire (0.075 mm in diameter). The inside diameter must be slightly larger than the outside diameter of the electron microscopy grid. The loop is then lowered onto the drop, the entire loop makes contact with the drop surface at the same time. This can be observed through drop deformation. So that no excess subphase is picked up, the loop should not go through the monolayer and into the subphase. The loop is then gently and carefully raised and lowered onto a glow-discharged grid. The grid is held with forceps and is parallel to the


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film in the loop. The transfer is made by hydrophilic contacts between the carbon foil and the crystal. The film is then broken by tilting the loop to increase the angle between the film and the grid. 10. In order to better evaluate the organizational state of the molecule in the crystallization experiment, it is useful to control its shape and size by direct adsorption of the sample on a carbon film and negative staining. Such an experiment will also give an insight into the aggregation state of the protein in solution. 11. The appearance of vesicular structures is often an indication for a too large excess of lipids. The working lipid solution should then be diluted. 12. To remove excess lipids, a detergent solution at low concentration can be used (19). Care must be taken during this step because the drop might migrate to both sides of the grid and interfere with the staining process.

References 1. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckmann, E., and Downing, K. H. (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol. Biol. 213, 899–929. 2. Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature 367, 614–621. 3. Amos, L. A., Henderson, R., and Unwin, P. N. (1982) Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog. Biophys. Mol. Biol. 39 , 183–231. 4. Uzgiris, E. E. and Kornberg, R. D. (1983) Two-dimensional crystallization technique for imaging macromolecules, with application to antigen-antibody-complement complexes. Nature 301, 125–129. 5. Darst, S. A., Ribi, H. O., Pierce, D. W., and Kornberg, R. D. (1988) Two-dimensional crystals of Escherichia coli RNA polymerase holoenzyme on positively charged lipid layers. J. Mol. Biol. 203, 269–273. 6. Schultz, P., Celia, H., Riva, M., Darst, S. A., Colin, P., Kornberg, R. D., et al. (1990) Structural study of the yeast RNA polymerase A. Electron microscopy of lipid-bound molecules and two-dimensional crystals. J. Mol. Biol. 216, 353–362. 7. Ribi, H. O., Reichard, P., and Kornberg, R. D. (1987) Two-dimensional crystals of enzyme-effector complexes: ribonucleotide reductase at 18-A resolution. Biochemistry 26, 7974–7979. 8. Lebeau, L., Regnier, E., Schultz, P., Wang, J. C., Mioskowski, C., and Oudet, P. (1990) Two-dimensional crystallization of DNA gyrase B subunit on specifically designed lipid monolayers. FEBS Lett. 267, 38–42. 9. Avila-Sakar, A. J., and Chiu, W. (1996) Visualization of beta-sheets and sidechain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography. Biophys. J. 70, 57–68. 10. Lebeau, L., Schultz, P., Celia, H., Mesini, P., Nuss, S., Klinger, C., et al. (1996) Specifically designed lipid assemblies as tools for two-dimensional crystallization of soluble biological macromolecules, in Handbook of Nonmedical Applica-

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tions of Liposomes, vol. II (Barenholz Y. and Lasic D. D., eds.), CRC, Boca Raton, FL, pp. 155–188. Darst, S. A., Ahlers, M., Meller, P. H., Kubalek, E. W., Blankenburg, R., Ribi, H. O., et al. (1991) Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules. Biophys. J. 59, 387–396. Kubalek, E. W., Le Grice, S. F., and Brown, P. O. (1994) Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid. J. Struct. Biol. 113, 117–123. Bischler, N., Balavoine, F., Milkereit, P., Tschochner, H., Mioskowski, C., and Schultz, P. (1998) Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids. Biophys. J. 74, 1522–1532. Mosser, G. and Brisson, A. (1991) Structural analysis of two-dimensional arrays of cholera toxin B- subunit. J. Electron Microsc. Tech. 18, 387–394. Kubalek, E. W., Kornberg, R. D., and Darst, S. A. (1991) Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy. Ultramicroscopy 35, 295–304. Fukami, A. and Adachi, K. (1965) A new method of preparation of a self-perforated micro plastic grid and its application. J. Electron Microscopy, 14, 112–118. Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W., et al. (1988) Cryo-electron microscopy of vitrified specimens. Quart. Rev. Biophys. 21, 129–228. Schmutz, M. and Brisson, A. (1996) Analysis of carbon film planarity by reflected light microscopy. Ultramicroscopy 63, 263–272. Asturias, F. J. and Kornberg, R. D. (1995) A novel method for transfer of twodimensional crystals from the air/water interface to specimen grids. EM sample preparation/lipid-layer crystallization. J. Struct. Biol. 114, 60–66.

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39 Atomic Force Microscopy of DNA and Protein-DNA Complexes Using Functionalized Mica Substrates Yuri L. Lyubchenko, Alexander A. Gall, and Luda S. Shlyakhtenko 1. Introduction Atomic force microscopy (AFM; also called scanning force microscopy [SFM]) is a rather novel technique that offers unique advantages in the potential for the very high resolution of DNA and small ligands in the absence of stains, shadows, and labels (1,2). Furthermore, the scanning can be performed in air or liquid. The latter is particularly important for resolving fully hydrated structures. The AFM is theoretically capable of resolving structural details at the level of atomic dimensions, provided that the specimen is not dynamic. A serious practical limitation to the application of AFM to structural and conformational studies of DNA and its complexes with proteins and other biological macromolecules has been sample preparation. The macromolecules must be tethered to the substrate surface in order to avoid resolution-limiting motion caused by the sweeping tip during scanning. Progress in sample preparation for AFM studies of DNA has been achieved in a number of groups (3–7) and some of these approaches have been applied to studies of a number of protein–DNA complexes (3,4,8). A versatile approach based on functionalization of surfaces with silanes was suggested in refs. 9–11. A weak cationic surface is obtained if aminopropyltrietoxy silane (APTES) is used to functionalize the mica surface with amine groups (AP-mica). This technique in addition to imaging nucleic acids under different conditions (10,12–14) was applied to imaging of a number of nucleoprotein complexes (9,11,15–17). Here, we describe a sample preparation procedure for AFM using AP-mica substrates. From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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The method of functionalization of mica is based on covalent attachment of 3-aminopropyltriethoxy silane to the surface of the mica, as shown schematically in Fig. 1. The amino groups of APTES are bound covalently to the freshly cleaved mica surface, giving it properties similar to an anion-exchange resin used in affinity chromatography. This group after being exposed to a water solution becomes positively charged in a rather broad range of pH (aliphatic amino groups have a pK of around 10.5). Therefore, DNA, which is a negatively charged polymer, should adhere to this surface strongly. The binding of DNA to AP-mica was monitored directly by the use of radiolabeled DNA. AFM imaging of AP-mica showed that very low concentration of APTES (less than 1 µM) should be used to obtain smooth surface (9,10). Vapor deposition of APTES allowed one to obtain the surface with mean roughness of several angstroms (9–12), so the DNA and DNA–protein complexes can be visualized easily (see Fig. 2A,B, respectively). The features of this procedure of sample preparation are as follows (9,11): • DNA binding to AP-mica is insensitive to the type of buffer and presence of Mg2+ or other divalent and miltivalent cations; hence sample preparation can be done in a variety of conditions. • Deposition can be done in a wide variety of pH and over a wide range of temperatures. • Once prepared, samples are stable and do not absorb any contaminants for months with minimal precautions for storing. • As low as 10 ng of DNA is sufficient for the preparation of one sample.

These characteristics of AP-mica were crucial for routine imaging nucleic acids (DNA, dsRNA, kinetoplast DNA) and nucleoprotein complexes of different type (9,16,17). 2. Materials. 1. Chemicals: commercially available 3-aminopropyltriethoxy silane (e.g., Fluka, Chemika-BioChemika (Switzerland), Aldrich (USA), United Chemical Technology (USA), and N,N-diisopropylethylamine (Aldrich, Sigma). It is recommended to redistill APTES and store under argon. 2. Mica substrate: any type of commercially available mica sheets (green or ruby mica). Asheville-Schoonmaker Mica Co. (Newport News, VA) supplies both thick and large (more than 5 × 7 cm) sheets suitable for making the substrates of different sizes. 3. Water: Double glass distilled or deionized water filtered through a 0.5-µm filter. 4. 2-L glass desiccators and vacuum line (50 mmHg is sufficient). 5. Plastic syringes (5–10 mL) with a plastic tip for rinsing the samples. 6. Plastic syringes (1 mL) for imaging in liquid. 7. Gas tank with clean argon gas. 8. Vacuum cabinet for storing the samples.


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Fig. 1. The reaction of aminopropyltriethoxy silane (APTES) with mica. Three possible types of reaction are illustrated.

3. Methods 3.1. AP-Mica Preparation 1. Place two plastic caps (cut them from regular 1.5 mL plastic tubes) on the bottom of a 2-L desiccator, evacuate then purge with argon. 2. Cleave mica sheets (approx 5 × 5 cm) to make them as thin as 0.1–0.05 mm and mount at the top of the desiccator. 3. Put 30 µL of APTES into one plastic cap in the desiccator and 10 µL of N,Ndiisopropylethylamine (Aldrich) into the other cap and allow the functionalization reaction to proceed for 1–2 h. Remove the cap with APTES and purge the desiccator with argon for 2 min. 4. Leave the sheets for 1–2 d in the desiccator to cure. The AP-mica is then ready for the sample deposition. (See Note 1.)

The procedure allows one to obtain a weak cationic surface with rather uniform charge distribution. This is illustrated in Fig. 2A by the uniform distribution of DNA fragments that can be obtained.

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3.2. Sample Preparation for AFM Imaging in Air 3.2.1. The Droplet Procedure 1. Prepare the solution of the sample (DNA, RNA, protein–DNA complex) in appropriate buffer. DNA concentration should be between 0.1 and 0.01 µg/mL, depending on the size of the molecules (see Notes 2 and 3). 2. Place 5–10 µL of the solution in the middle of AP-mica substrate (usually 1 × 1-cm squares) for 2–3 min. 3. Rinse the surface thoroughly with water (2–3 mL per sample) to remove all buffer components. A 10-mL plastic syringe is very useful for rinsing, but attach an appropriate plastic tip instead of a metal needle. 4. Dry the sample by blowing with clean argon gas. The sample is ready for imaging. Store the samples in vacuum cabinets or desiccators filled with argon.

3.2.2. The Immersion Procedure This procedure is recommended if the deposition should be performed at strictly controlled temperature conditions (0°C or elevated temperatures). 1. Prepare the solution (DNA, RNA, nucleoprotein complexes) and preincubate for 10–20 min to allow the temperature to equilibrate. Recommended concentration of DNA is 0.2 –0.01 µg/mL, depending on the size of the molecules (see Notes 2 and 3). 2. Immerse a piece of AP-mica into the vials and leave it for 10–20 min to allow the samples to adsorb to the surface. 3. Remove the specimen, rinse with water thoroughly, and dry under the argon flow. The sample is ready for imaging. 4. The samples can be stored in vacuum cabinet or under argon.

3.3. AFM Imaging in Air 1. Mount the sample and start approaching the probe. 2. Both the contact and intermittent (tapping) modes can be used, but the latter is preferable and allows one to obtain images of DNA and DNA–protein complexes routinely. Our experience is mostly limited to a NanoScope III microscope (MultiMode system, Digital Instruments, CA), but samples prepared on AP-mica were imaged on other commercially available instruments (e.g., the microscopes manufactured by Topometrix, Park Scientific Instruments, Molecular Imaging). With the MultiMode system, any type of probe designed for noncontact imaging can be used. NanoProbe TESP tips (Digital Instruments, Inc.) and conical sharp Fig. 2. (previous page) AFM images of a 800-bp fragment (A) and reconstituted chromatin (B). The concentration of DNA was 0.5 µg/mL in (A). Reconstituted chromatin was deposited onto the substrate after glutaraldehyde fixation. (The sample was from D. Lohr [Arizona State University] and the images were taken in air with TM AFM [NanoScope III]).

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Lyubchenko et al. silicon probes from K-TEK International (Portland, OR) work well. Typically a tapping frequency of 240–380 kHz and a scanning rate 2–3 Hz allows one to obtain stable images.

3.4. Imaging in Solution The capability of AFM to perform scanning in liquid is its most attractive feature for numerous biological applications, allowing imaging under nearphysiological conditions. In addition, this mode of imaging permits one to eliminate the undesirable resolution-limiting capillary effect typical for imaging in air (3,8,9). As a results, images of DNA filaments as thin as approx 3 nm were obtained in water solutions (13) and helical periodicity was observed when dried DNA samples were imaged in propanol (18). AP-mica can be used as a substrate for imaging in liquid. Moreover, the first images of DNA in fully hydrated state were obtained by the use of AP-mica (19). This section describes the procedures of two types of imaging in solution.

3.4.1. Imaging of Dried Sample in Solutions This type of imaging was successfully applied for high-resolution imaging of DNA (18,19). 1. Install an appropriate tip designed for imaging in liquid (fluid cell). Use stiff triangular Si3Ni4 cantilevers (20). 2. Mount the sample on the stage of the microscope. Coating the stage of the scanner with a thin plastic film prevents it from being wetted because of accidental leakage of the fluid beneath the mica sheet. 3. Attach the head of the microscope with installed fluid cell and make appropriate adjustments to the microscope. 4. Approach the sample to the tip manually, leaving approx 20-µm gap between the tip and the surface. 5. Inject buffer solution or appropriate solvent with a 1-mL plastic syringe through the inlet hole in the fluid cell. 6. Change the position of the mirror to maximize the signal on the photodetector. 7. Find a resonance peak. Typically, it is quite broad peak around 8–9 kHz for the MultiMode system. Follow the recommendations given in the manual for the fluid cell on how to find the peak. 8. Minimize the drive amplitude. The numbers vary from tip to tip, but amplitudes as low as 10 nm or even less provide better quality pictures (see Note 4). 9. Allow the microscope to approach the sample and engage the surface. 10. Operate with the setpoint voltage and drive the amplitude parameters to improve the quality of images (see Note 4).

3.4.2. Imaging Without Drying of the Sample (AFM In Situ) This type of imaging is recommended in cases in which dynamics are to be studied.


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1. Prepare the solution of your sample in appropriate buffer. The concentration is the same as it is needed for imaging in air (Subheading 3.2.) 2. After installing the tip in the fluid cell, mount a piece of AP-mica on the sample stage. Mica pieces of 1 cm × 1 cm are sufficient for NanoScope design of the fluid cell. As earlier, coating the stage with a plastic film is recommended for secure operating of the microscope. 3. Take approx 50 µL of the sample with a 1-mL syringe. Use 200-µL plastic tips with capillary ends instead of a metal needle. Cut both ends of the tip to fit the syringe and the diameter of inlet hole of the fluid cell. 4. After necessary adjustments of the microscope (Subheading 3.4.1.) and manual approaching of the tip, inject the solution into the cell. The use of additional syringe attached to the outlet of fluid cell as a suction helps in manipulating the small volume of solution. 5. Start approaching and follow the steps described in Subheading 3.4.1.

3.5. Alternative Procedures for AFM Sample Preparation Among other techniques applied to AFM studies of DNA, the method based on miltivalent cations (3,5,8) has permitted the imaging of a number of nucleoprotein complexes (3,4,8). In this approach, the mica surface is simply treated with multivalent ions (e.g., Mg2+) to increase its affinity for DNA, the DNA then being held in place strongly enough to permit reliable imaging by AFM. An alternative is to deposit the sample in the buffer containing a multivalent ion. This cation-assisted procedure of sample preparation was used for studies of the process of DNA degradation with nuclease (21) and interaction of DNA with photolyase (22). The mechanism underlying this technique remains unclear and the protocol depends on the system studied and the type of the cation used, and the efficiency of DNA deposition is buffer sensitive (23,24). In some cases, a special type of tips (electron-beam-deposited tips) is required for reliable imaging (25). A protocol describing the use of Mg-assisted procedure has been published (26). 4. Notes 1. A dry argon atmosphere is crucial for obtaining the substrates for AFM studies and for storage of the substrate. Allow the gas to flow while desiccator is opened. With these precautions, the AP-mica substrates retain their activity for several weeks. 2. DNA concentration. This parameter depends on the length of molecules. If the molecules are as small (e.g., several hundred base pairs), a concentration of approx 0.3 µg/mL is recommended to avoid intermolecular crossing. A lower DNA concentration is recommended for larger DNA molecules. For example, concentration of lambda DNA (approx 48 kb) of approx 0.01 µg/mL allows one to obtain images of individual DNA molecules (9–11,19). 3. DNA preparation. Very little DNA is needed to prepare the samples by the droplet procedure. Typically, 10 ng of DNA is sufficient for the preparation of plas-

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mid DNA (approx 3 kb long). Because a band of DNA from agarose gel usually contains 100 ng of DNA, DNA extracted from a single gel slot can be sufficient for preparation of a complete set of samples. The following procedures can be used for purification of DNA extracted from the gel: • Electrophoretic deposition of DNA bands onto DEAE paper. Strips of DEAE paper are placed into a slot cut in the agarose gel 3–5 mm below the band to be recovered and the DNA is electrophoresed onto the paper for 5–10 min (the time can be determined by direct examination the gel under an ultraviolet [UV] source). The DNA is extracted from the paper by elution into 2 M NaCl followed by two rounds of spin-column desalting and extensive ethanol precipitation. • Extraction from the gel. The procedure is based on the use of the extraction kit UltraClean15 (MoBio Laboratories, Solana Beach, CA). The purification consists of melting of the slice of agarose, immobilizing the DNA on the absorbent matrix, washing off all contaminants, and eluting DNA from the matrix with a low-salt buffer. At least one step of the ethanol precipitation is needed to remove UV absorbing low-molecular-weight materials. A similar procedure can be applied to purification of the sample eluted from polyacrylamide gel. 4. Imaging conditions. It was recommended to operate the instrument at the lowest possible drive amplitude. This recommendation is based on the following considerations; the oscillating tip provides rather large energy to the sample. According to ref. 45, a total energy provided to the sample by oscillating tip can be as high as 10–16–10–17 J at 30 nm amplitude of oscillation. However, this value is almost three orders of magnitude lower if the microscope is operated at an amplitude as low as approx 3 nm. Such imaging conditions allow one to minimize the effect of the tip on the sample, to prevent damaging the tip, and to obtain images with high contrast. In addition, such conditions simplify considerably the study by AFM of dynamic processes such as segmental DNA mobility (13,14) or the process of protein–DNA interaction (27).

Acknowledgments This work was supported by grant GM 54991 from the NIH. References 1. Binnig, G., Quate, C. F., and Gerber, C. H. (1986) Atomic force microscope Phys. Rev. Lett. 56, 930–933. 2. Hansma, P. K., Elings, V. B., Marti, O., and Bracker, C. E. (1988) Scanning tunneling microscopy and atomic force microscopy: some applications to biology and technology. Science 242, 209–216. 3. Bustamante, C., Erie, D. A., and Keller, D. (1994) Biochemical and structural applications of scanning force microscopy. Curr. Opin. Struct. Biol. 3, 750–760. 4. Bustamante, C. and Rivetti, C. (1996) Visualizing protein-nucleic acid interactions on a large scale with scanning force microscope. Annu. Rev. Biophys. Biomol. Struct. 25, 395–429.


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5. Vesenka, J., Guthold, M., Tang, C. L., Keller, D., and Bustamante, C. (1992) Substrate preparation for reliable imaging of DNA molecules with the scanning force microscopy. Ultramicroscopy 42–44, 1243–1249. 6. Yang, J., Takeyasu, K., and Shao, Z. (1992) Atomic force microscopy of DNA molecules. FEBS Lett. 301, 173–176. 7. Allen, M. J., Dong, X. F., O’Neil, T. E., Yau, P., Kowalczykowski, S. C., Gatewood, J., Balhorn, R., et al. (1993) Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences. Biochemistry 32, 8390–8396. 8. Hansma, H., G., and Hoh, J. (1994) Biomolecular imaging with the atomic force microscopy. Ann. Rev. Biophys. Biochem. Struct. 23, 115–139. 9. Lyubchenko Y. L., Jacobs B. L., Lindsay S. M., and Stasiak A. (1995) Atomic force microscopy of protein–DNA complexes [Review]. Scanning Microsc. 9, 705–727. 10. Lyubchenko, Y. L., Gall, A. A., Shlyakhtenko, L. S., Harrington, R. E., Oden, P. I., Jacobs, B. L., et al. (1992) Atomic force microscopy imaging of double stranded DNA and RNA J. Biomolec. Struct. Dynam. 9, 589–606. 11. Lyubchenko, Y. L., Blankenship, R. E., Lindsay, S. M., Simpson, L., Shlyakhtenko, L. S. (1996) AFM studies of nucleic acids, nucleoproteins and cellular complexes: The use of functionalized substrates. Scanning Microsc. 10(Suppl.), 97–109. 12. Lyubchenko, Y. L., Jacobs, B. L., and Lindsay, S. M. (1992) Atomic force microscopy imaging of reovirus dsRNA: a routine technique for length measurements. Nucleic Acids Res. 20, 3983–3986. 13. Lyubchenko Y. L. and Shlyakhtenko, L. S. (1997) Visualization of supercoiled DNA with atomic force microscopyin situ. Proc. Natl. Acad. Sci. USA 94, 496–501. 14. Shlyakhtenko, L. S, Potaman, V. N., Sinden, R. R., and Lyubchenko, Y. L. (1998) Structure and dynamics of supercoil-stabilized DNA cruciform. J. Mol. Biol. 280, 61–72. 15. Lyubchenko, Y. L., Oden, P. I., Lampner, D., Lindsay, S. M., and Dunker, K. (1993) Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces, Nucleic Acids Res. 21, 1117–1123. 16. Lyubchenko, Y. L., Shlyakhtenko, L. S., Aki, T., and Adhya, S. (1997) AFM visualization of GalR mediated DNA looping. Nucleic Acids Res. 25, 873–876. 17. Herbert A., Schade, M., Lowenkaupt, K., Alfken, J., Schwartz, T., Shlyakhtenko, L. S., et al. (1998) The Za domain from human ADAR1 binds to the Z-DNA conformer of many different sequences. Nucleic Acid Res. 26, 3486–3493. 18. Hansma, H. G., Laney, D. E., Bezanilla, M., Sinsheimer, R. L., and Hansma, P. K. (1995) Applications for atomic force microscopy of DNA. Biophys. J. 68, 672–1677. 19. Lyubchenko, Y. L., Shlyakhtenko, L. S., Harrington, R. E., Oden, P. I., and Lindsay, S. M. (1993) AFM imaging of long DNA in air and under water. Proc. Natl. Acad. Sci. USA 90, 2137–2140. 20. Hansma, P. K., Cleveland, J. P., Radmacher, M., Walters, D. A., and Hillner, P. (1994) Tapping mode atomic force microscopy in liquids. Appl. Phys. Lett. 64, 1738–1740.

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21. Bezanilla, M., Drake, B., Nudler, E., Kashlev, M., Hansma, P. K., and Hansma, H. G. (1994 Motion and enzymatic degradation of DNA in the atomic force microscope Biophys. J. 67, 2454–2459. 22. Han, W. H., Lindsay, S. M., and Jing, T. W (1996) A magnetically-driven oscillating probe microscope for operation in liquids. Appl. Phys. Lett. 69, 4111–4114. 23. Bezanilla, M., Manne, S., Laney, D. E., Lyubchenko, Y. L., and Hansma, H. G (1995) Adsorption of DNA to mica, silylated mica, and minerals: characterization by atomic force microscopy. Langmuir 11, 655–659. 24. Hansma, H. G. (1996) A useful buffer for atomic force microscopy of DNA. Sci. Tools Pharmacia Biotech. 1(3), 7. 25. Kasas, S., Thomson, N. H., Smith, B. L., Hansma, H. G., Zhu, X., Guthold, M., et al. (1997) Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry 36, 461–468. 26. Hansma, H. G. (1998) Atomic force microscopy of DNA on mica in air and fluid, in Procedures for Scanning Probe Microscope (Colton, R. J., et al., eds.), Wiley, Chichester, pp. 389–393. 27. van Noort, S. J. T. , van der Werf, K. O., Eker, A. P. M., Wyman, C., Grooth, B. G., van Hulst, N. F., et al. (1998) Direct visualization of dynamic protein–DNA interactions with a dedicated atomic force microscope. Biophys. J. 74, 2840–2849.

Additional Reading Colton et al., (eds.) (1998) Procedures in Scanning Probe Microscopes. Wiley, Chichester.

Electron Microscopy of Nucleoprotein Complexes

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40 Electron Microscopy of Protein–Nucleic Acid Complexes Uniform Spreading of Flexible Complexes, Staining with a Uniform Thin Layer of Uranyl Acetate, and Determining Helix Handedness Carla W. Gray 1. Introduction There are a number of proteins involved in DNA replication, recombination, or repair that bind stoichiometrically to single DNA strands irrespective of the nucleotide sequence, and some of these proteins also bind to singlestranded RNA. Some of the best known examples are the ssb protein of Escherichia coli, the gene 32 protein of phage T4, the gene 5 protein of the M13/fd/f1 filamentous bacterial viruses, and the more recently isolated human replication protein A (1–4). Complexes formed by these proteins contain protein bound to the nucleic acid at defined ratios of the number of nucleotides per molecule of bound protein; the ratios are determined by the interactive properties of the protein. These ratios, and the structures of the complexes that are formed, may vary with factors such as changes in solution conditions that alter the binding properties of the proteins. Stoichiometric, multiprotein complexes of proteins with nucleic acids will form structural repeats, arranged as discrete clusters of bound proteins or as a continuous nucleoprotein helix. Although the individual proteins may not be resolved, the structural repeats tend to be of a size that can be visualized by electron microscopy. “Negative” staining, in which protein masses are delineated by their exclusion of an electron-opaque stain, is a method of choice because negative staining provides a well-contrasted image at higher resolution than is attained with other techniques such as shadowing with refractory From: Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed. Edited by: T. Moss © Humana Press Inc., Totowa, NJ

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Fig. 1. A transmission electron micrograph of a helical complex of the fd gene 5 protein with circular, single-stranded fd viral DNA. The complex was formed in vitro and was spread and stained with uranyl acetate by the methods described in this chapter. The complex is not tilted; the plane of the support film is in the plane of the page.

metals. We have used negatively stained complexes with the M13/fd/f1 gene 5 protein to provide crucial information in conjuntion with crystallographic and low-angle X-ray scattering studies, making it possible to model the threedimensional structures of the complexes (5,6). Negatively stained nucleoprotein complexes can be prepared relatively quickly and examined immediately after preparation. This makes it advantageous to use negatively stained preparations prior to or in conjunction with cryo electron microscopy, a technique that, although it offers better preservation for detailed structural studies, is a more difficult and time-consuming method. Nucleoprotein complexes are often highly flexible and the complexes are easily distorted, tangled, or partially dissociated during preparation for negative staining. The author has developed procedures to overcome these difficulties (3,7), such that preparations consistently contain complexes with well-extended configurations free of any obvious distortions (Fig. 1). Complexes prepared in this manner are uniformly spread on a two-dimensional support film and can be used for quantitative analysis of such parameters as the number of protein clusters or helical turns in a complex, the axial length of the complex, and the extent of the local variations in interturn distances in a complex that forms a flexible helix. These preparations can also be used for analyses of three-dimensional structures using tilted specimens (3). The most likely

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Fig. 2. Effects of tilting on the appearance of left- and right-handed helices. Threedimensional images are drawn in the upper panel of the figure and two-dimensional parallel projections in the lower panel. A negatively stained complex will be seen as a two-dimensional projection. A helix at 0° tilt is parallel to the plane of the page; a “plus” (+) tilt brings the top of the helix nearer to the observer, whereas a “minus” (–) tilt brings the bottom of the helix closer to the observer. (Reprinted with permission from ref. 3.)

application is a determination of the handedness of helical nucleoprotein complexes, using the approach of Finch (8), which is illustrated for a general case in Fig. 2. Projections of left- or right-handed helices on a two-dimensional plane are identical, as shown in the lower part of the figure. However, a lefthanded helix tilted with its top toward the viewer will show deeper indentations between the helical turns along its right side, whereas an identically tilted right-handed helix has the deeper indentations on its left side. Tilting the helices in the opposite direction, with the tops of the helices away from the viewer, produces an opposite set of left-hand and right-side indentations. In the description that follows, we describe a practical means of determining the absolute orientations of helices in images of tilted specimens. 2. Materials 1. Purified water, chemically softened and then predistilled in bulk, is twice redistilled in our laboratory using a series of two 24-in. borosilicate glass Vigreux columns. We do not find it necessary to use a quartz still. Alternatively, the bulk distilled water may be deionized to a resistivity of 18 mΩ·cm in a Millipore MilliQ system consisting of one Milligard cellulose ester prefilter cartridge, two ion-

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3.

4.

5.

6.

Gray exchange cartridges, and a 0.22-µm filter, in series. No activated-charcoal filter is included in our system, because of the tendency of the charcoal filter to release minute charcoal particles, which are found in our preparations for electron microscopy. (See Note 1.) Buffers in which the protein–DNA complexes are to be visualized. Concentrated buffer stocks are passed through a 0.22-µm fiberless polycarbonate filter (Nuclepore/Costar) to remove particulates and are then stored in borosilicate glass or polystyrene containers. The buffers should generally not contain high concentrations of salts or other nonvolatile components, as these components will tend to be retained on the carbon support film and can interfere with visualization of the protein–DNA complexes. The protein–nucleic acid complexes are to be examined. These preparations should not contain significant excess quantities of noncomplexed or contaminating proteins, and they must generally be free of contaminating nonaqueous solvents as well as lipids, oils, salts, detergents, and other nonvolatile materials. We frequently repurify proteins and nucleic acids obtained commercially or from other laboratories, using ethanol precipitation of nucleic acids, molecular-sieve chromatography of proteins, or dialysis. About 0.2–2 nmol of DNA (measured as the concentration of nucleotides), together with protein added at an appropriate ratio, will be needed for a 50-µL incubation mixture from which the protein– DNA complexes are adsorbed to a single specimen grid. Glutaraldehyde, purified for electron microscopy (distilled in vacuo and stored in sealed ampoules under inert gas). The contents of one ampoule are diluted to 8% (v/v) in purified water and are stored at –20°C in a tightly capped borosilicate glass tube with a Teflon-lined screw cap; this solution can be used for as long as 6 mo. A suitable electron-opaque (“negative”) stain, preferably analytical reagent grade. We generally use uranyl acetate, which provides good surface detail and yields satisfactory images with many proteins. However, one should always consider the possibility that other stains may be preferable for a particular application (9). A 2% (w/v) solution of a small amount of uranyl acetate in purified water is dissolved by stirring for 30 min in a borosilicate glass beaker. The beaker is sealed with a wax film and stored in the dark. The solution is used within a few days or weeks, but only if precipitates have not begun to form. Uranyl salts are weakly radioactive, and discarded solutions should be collected and properly disposed of as radioactive waste. Carbon support films, 8–10 nm thick, on 500-mesh copper grids. We make our films in an Edwards E306A evaporator equipped with a liquid-nitrogen trap and a quartz crystal film thickness monitor (Edwards FTM5). The chamber is evacuated just to 1 × 10–4 mbar, contaminants are burnt off from the carbon rods (shutter closed, carbon rods brought to a red glow) and then evaporation is carried out over a period of several seconds. We use high-purity carbon rods (Bio-Rad/ Polaron, CG>TA

Comments

Uses

Does not attack Z-form DNA and more weakly (35%) attacks A form DNA as compared to B form.

Footprinting.

Out of plane attack on bases

DNA conformation, base destacking, bending.

DEPC

162

G + A ¨ C, unstacked bases

Dimethyl Sulphate (DMS)

126

N7-G via major groove N3-A via minor groove

DNase I

40k

Independent attack on phosphodiester bonds from minor groove face of DNA

Sequence specific

Footprinting Interference

Footprinting.

DNA cleavage

Ethyl-nitroso-urea

117

60–65% Non-esterified oxygen of phosphodiester (> T-O2 (minor groove) =G-O6 (major groove) > T-O4 (major groove) >> C-O2 (minor groove))

Low sequence specificity

Interference

Exonuclease III

28k

3 terminal attack

Extremities of DNA-protein complexes

“Footprinting”

Continued...

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Appendix II

Hydroxy-radical

17

Sugar backbone

Little sequence preference

Footprinting Interference

Osmium tetroxide

254

Unstacked T

Very specific for certain, as yet poorly defined DNA conformations including overwinding

DNA conformation

Permanganate

119

T » G, C, A

Out of plane attack on 5,6 double bond of T

DNA conformation, melting, base destacking, bending.

Diffusible singlet oxygen

16

Unstaked/unwound DNA bases

DNA conformation

uranyl(VI) ion (UO22+)

268

Phosphates-photolysis

Little sequence preference in duplex DNA (though as free bases, G is by far the prefered target) Little sequence dependence

—

TT >> CT, TC, or CC

UV irradiation

Footprinting of both macromolecules and drugs.

Products: Footprinting. i) cyclobutane Note, protein pyrimidine does not dimer (CBD). prevent ii) pyrimidine (6-4) access but pyrimidone changes DNA photoproduct reactivity (6-4PP) locally.

Index

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Index A AFM, see Atomic force microscopy Aminopropyltrietoxy silane (APTES), mica functionalization, see Atomic force microscopy 1-Anilinonaphthalene-8-sulfonic acid (ANS), competition assay for DNA binding, advantages over intrinsic fluorescence, 265 binding curve generation, 270–272 materials, 267, 268, 273 preliminary testing, 266, 267, 269, 270, 273 principle, 265 resonance energy transfer, 273, 274 titration, 268, 269, 273 protein binding properties, 265, 266 ANS, see 1-Anilinonaphthalene-8sulfonic acid APTES, see Aminopropyltrietoxy silane Atomic force microscopy (AFM), advantages and limitations, 569 functionalized mica substrates, overview, 569, 570 preparation, 571, 575 imaging in air, contact vs tapping modes, 569, 570 sample preparation, droplet procedure, 569, 571, 572 immersion procedure, 569, 571, 572 imaging in solution,

advantages, 570 dried samples, 570, 572 in situ, 570, 571 materials for nucleoprotein imaging, 570 multivalent cation method for sample preparation, 571 8-Azidoadenine, DNA incorporation, materials, 325, 326 nick translation, 328, 332 DNA–protein crosslinking, filter binding assay, 330 gel electrophoresis analysis, 329, 330 photocrosslinking, 328, 329, 332, 333 photoaffinity labeling overview, 323, 324 synthesis, characterization, 327, 328, 331, 332 materials, 325 protocol, 326, 327, 330, 331 radiolabeled compound, 330 B Biacore, see Surface plasmon resonance C Calorimetry, see also Differential scanning calorimetry; Isothermal titration calorimetry, DNA-binding protein applications, 512

621

622 equilibrium, 513 thermodynamic parameters, energetics of protein–DNA interaction, 512, 513 overview, 511, 512 CD, see Circular dichroism Circular dichroism (CD), applications for DNA-binding proteins, 504, 508 DNA change measurement upon protein binding, materials, 504, 505 titration, 505, 506, 508 wavelength selection, 504, 505 origin of signal, 503 principles, 503, 504 sensitivity, 504 Sox-5 HMG domain dissociation constant determination for DNA, 518 Crystallography, see Reconstitution, protein–DNA complexes for crystallization; Two-dimensional crystallization D DEPC footprinting, see Diethyl pyrocarbonate footprinting Diethyl pyrocarbonate (DEPC) footprinting, advantages, 63, 64 applications, 64, 65 DNA modification, detection of modified bases with piperidine cleavage, 65, 66, 68, 69, 71 reaction mechanism, 64 in vitro experiments on linear DNA fragments, binding reaction, 68, 71 gel electrophoresis, 69, 71 modification reaction and stopping, 68, 71

Index piperidine cleavage, 68, 69, 71 radiolabeling of probe, 68 materials, 67 Differential scanning calorimetry (DSC), see also Calorimetry, excess molar heat capacity, 516 intrinsic heat capacity, 516 partial molar heat capacity, 515, 516 principle, 515, 516 Sox-5 HMG domain interaction with DNA, concentration determinations, complex, 528, 529 DNA, 527, 5286 protein, 528 correction of isothermal titration calorimetry-derived enthalpies, 525, 526 data acquisition, 524, 530, 531 data analysis, 525 instrumentation, 520 materials, 520, 521, 529 Dimethyl sulfate (DMS) footprinting, combination with electrophoretic mobility shift assay, 78, 79 ligation-mediated polymerase chain reaction for in vivo footprinting, advantages and limitations, 183, 187 cleavage for DNA sequencing products, A reaction, 201 C reaction, 201, 202 G reaction, 201 overview, 200, 201 processing of samples, 202 reagants, 192 T+C reaction, 201 detectable DNA–protein interactions, 183 dimethyl sulfate treatment, 193, 202, 203, 213 DNA polymerase selection, 191

Index DNA purification, DNA extraction, 200, 213 materials, 191, 192 nuclei isolation, 200 quantification, 200, 213 gel electrophoresis and electroblotting, blotting, 209, 214 electrophoresis, 208, 209 materials, 196, 197 hybridization, digoxigenin-labeled probe, 197, 198, 210, 214, 215 materials, 197 radiolabeled probe, 197, 209, 214 ligation, ligation reaction, 207 materials, 195 modified bases and conversion to single-strand breaks, 181, 188, 193, 205 overview, 176, 177 polymerase chain reaction, cycles, 207, 208 materials, 195, 196, 213 primer extension, incubation conditions, 206, 207 materials, 194, 195, 212, 213 single-stranded hybridization probe preparation, amplification product purification and quantification, 198, 199, 211, 215 digoxigenin labeling, 199, 212, 215 length, 210, 215 materials, 198, 199, 213 polymerase chain reaction amplification, 198, 210, 211 radiolabeling, 199, 211, 212

623 methylation interference assay, see methylation protection/ interference, dimethyl sulfate principle, 78 sites of reaction with DNA, 79 DMS footprinting, see Dimethyl sulfate footprinting DNA bending, functions, 403 pBend vectors for assay, bending angle, 415, 416 cloning sites, 405 colony screening for clones, 408, 411 electrophoretic analysis of complexes, 408, 409, 412–414 host strains of bacteria, 409 materials, 405–409 overview, 403, 404 protein-binding site insertion into plasmid, 405–411, 413 purification of plasmid DNA, 408, 411, 412 restriction sites, 404–406, 414, 415 types of vectors, 404, 405 DNase I footprinting, applications, 31, 32 autoradiography, 36, 38 binding reaction, 35–37 combination with electrophoretic mobility shift assay, 78, 79 digestion, 36, 37 DNA probe labeling, 34 DNase I, sequence specificity, 33, 34, 79 structure and function, 32, 33, 79 gel electrophoresis, 36, 37 ligation-mediated polymerase chain reaction for in vivo footprinting, advantages and limitations, 186, 187 cleavage for DNA sequencing products, A reaction, 201

624

Index C reaction, 201, 202 G reaction, 201 overview, 200, 201 processing of samples, 202 reagants, 192 T+C reaction, 201 DNA polymerase selection, 191 DNA purification, DNA extraction, 200, 213 materials, 191, 192 nuclei isolation, 200 quantification, 200, 213 DNase I treatment, 193, 204, 205, 213, 214 gel electrophoresis and electroblotting, blotting, 209, 214 electrophoresis, 208, 209 materials, 196, 197 hybridization, digoxigenin-labeled probe, 197, 198, 210, 214, 215 materials, 197 radiolabeled probe, 197, 209, 214 ligation, ligation reaction, 207 materials, 195 modified bases and conversion to single-strand breaks, 188 nonspecific priming of 3'-ends, 188, 189 overview, 182, 186, 188 permeabilization of cells, 188 polymerase chain reaction, cycles, 207, 208 materials, 195, 196, 213 primer extension, incubation conditions, 206, 207 materials, 194, 195, 212, 213 single-stranded hybridization probe preparation,

amplification product purification and quantification, 198, 199, 211, 215 digoxigenin labeling, 199, 212, 215 length, 210, 215 materials, 198, 199, 213 polymerase chain reaction amplification, 198, 210, 211 radiolabeling, 199, 211, 212 materials, 35–37 nonspecific competitor DNA, 34 principle, 31, 77, 78 Southwestern blotting, see Southwestern blot titration, 34 DSC, see Differential scanning calorimetry E Electron microscopy, see also Scanning transmission electron microscopy, nucleoprotein structural repeat imaging, focusing effects, 584, 585 materials, 571–573, 586 negative staining, 579, 580, 583, 584 overview, 579 specimen mounting, 583, 586, 587 tilted specimens, applications, 580, 581 closer end identification, 584–586 Fresnel patterns, 585, 586 helical asymmetry identification, 584–586 two-dimensional crystallography, see also Two-dimensional crystallization, crystal transfer to grid, 563, 565–567

Index evaluation of specimens, 564, 567 negative staining, 563, 567 support preparation, 616–614 Electrophoretic mobility shift assay (EMSA), advantages, 13 applications, 15, 16 buffers, 20, 26 combination with binding interference studies, 16 DNA probe, isolation, double-stranded synthetic oligonucleotides, 23, 27 fragments derived from subcloned sequence, 22, 23 materials, 20, 21 labeling options, 19 radiolabeling, double-stranded synthetic oligonucleotides, 22 fragments derived from subcloned sequence, 21, 22, 27 materials, 20, 25 safety, 25 size, 19 electrophoresis, autoradiography, 24 gel matrix selection, 19, 20, 25, 26 gel preparation, 23, 24, 26 loading and electrophoresis, 24, 26 materials, 21, 25, 26 ethylation interference assay, 233–236, 240, 241 exonuclease III footprinting optimization, 43, 45, 46 hydroxyl radical footprinting optimization, 55 hydroxyl radical interference, gel, 250, 252 optimization, 250, 252 10-Phenanthroline-copper footprinting coupling,

625 benefits, 83, 85, 86 cleavage in gel, 94, 95, 106, 107 competition binding assay, 92, 104 dissociation rate determination, 92, 104, 105 gel preparation, 93 loading of gel, 94, 106 materials, 86, 87 optimization, binding reaction parameters, 103, 104 electrophoresis conditions, 104 exposure time to chemical nuclease, 92, 105, 106 preliminary assay, 92, 102 probe length, 103 preparative reaction, 93 principle, 82 running conditions, 94 reconstitution of protein–DNA complexes for crystallization, 551, 554 restriction endonuclease dissociation constant determination, competitive equilibrum binding, 482 data analysis, 482, 383 direct titration, 481, 482, 487, 488 materials, 471 sensitivity, 13 two-wavelength femtosecond laser crosslinking optimization, 614, 615 ELISA, see Enzyme-linked immunosorbent assay EMSA, see Electrophoretic mobility shift assay Enzyme-linked immunosorbent assay (ELISA), phage binding, 419, 423, 424, 428 Equilibrium constant, filter-binding assay determination, 5, 6

626 fluorescence anisotropy determination, 469, 486, 487 Ethylation interference, ethylnitrosourea, modification of phosphate groups, 229 modification reaction, 233, 235, 239 secondary modifications, 229, 239 fractionation of DNA by electrophoretic mobility shift assay, 233–236, 240, 241 materials, 230, 231, 233, 234 MetJ methionine repressor interaction with target DNA, 230, 237–239 phosphotriester cleavage, 236 principle, 230 radiolabeling of DNA, 230, 231, 233–235 recovery of DNA from gels, 236 sequencing of DNA, 234, 236, 237, 241, 242 Exonuclease III footprinting, applications, 41 digestion reaction, 43–46 exonuclease III, activities, 39 sequence specificity, 39 gels, band-shift assay, 42 electrophoresis, 44 purification of binding complexes, 44, 45 sequencing, 42 interpretation, 40 materials, 42 optimization using electrophoretic mobility shift assay, 43, 45, 46 principle, 40 F Filter-binding assay,

Index advantages, 1 buffers, 3 equilibrium constant determination, 5, 6 equipment, 4 filters, 3 in vitro selection, 9, 10 kinetic measurements, association, 7 dissociation, 6, 7 interference measurements, 7, 8 methionine repressor binding to operator variants, 8, 9 radiolabeling of DNA, gel electrophoresis and band excision, 4 labeling reaction, 4 materials, 2, 3 plasmid digestion, 4 restriction endonuclease dissociation constant determination, competitive equilibrium binding, 478–480, 487, 484 data analysis, competitive titration, 481 direct binding, 478, 485, 486 direct titrations, 476, 477, 485–487 materials, 471 retention efficiency, 2 troubleshooting, 10 Fluorescence anisotropy, DNA–protein dissociation constant determination, 493 equilibrium constant determination, 469, 486, 487 restriction endonuclease dissociation constant determination using hexachlorofluorescein-labeled oligonucleotides, data analysis, 483, 484, 488 fluorescence measurements, 483, 488 materials, 470–472 overview, 469, 470, 486, 487

Index Footprinting, see Diethyl pyrocarbonate footprinting; Dimethyl sulfate footprinting; DNase I footprinting; Exonuclease III footprinting; Hydroxyl radical footprinting; In vivo DNA footprinting; Osmium tetroxide footprinting; 1,10-Phenanthrolinecopper footprinting; Potassium permanganate footprinting; Singlet oxygen footprinting; Ultraviolet C footprinting; Ultraviolet-laser footprinting; Uranyl photofootprinting G Gel retardation assay, see Electrophoretic mobility shift assay H Histone, see Linker histone-Fe(II) EDTA conjugate; Ultraviolet laser-induced protein–DNA crosslinking Hydroxyl radical footprinting, advantages, 49 applications, antibiotic–DNA complexes, 54 DNA structure probing, 54 protein–DNA complexes, 53, 54 RNA–protein complexes, 54 RNA structure probing, 54 binding reaction, 56, 57, 59 cutting reaction, materials, 54, 58 mechanism, 50 optimization, 56, 59 DNA probe preparation and labeling, 56, 59 gels, nondenaturing, 55

627 sequencing, 55, 58, 59 generation of radicals, 49, 50 interpretation, 51–53 optimization using electrophoretic mobility shift assay, 55 principle, 49–51 separation of free DNA from complex, nitrocellulose filter filtration, 58, 59 nondenaturing gel electrophoresis, 57–59 Hydroxyl radical interference, advantages, 245, 246 applications, RNA polymerase–promoter interaction, 248, 249 transcription factors, 247, 248 cutting reaction, 249–252 electrophoretic mobility shift assay, gel, 250, 252 optimization, 250, 252 generation of radicals, 246 interpretation, 246 materials, 249, 250, 252 principle, 245, 246 sequencing, 249–252 Hydroxyl radical site-directed cleavage, see Linker histone-Fe(II) EDTA conjugate I 1,5-IAEDANS, competition assay for DNA binding, 274 In vivo DNA footprinting, dimethyl sulfate footprinting, see Dimethyl sulfate footprinting DNase I footprinting, see DNase I footprinting osmium tetroxide footprinting, see Osmium tetroxide footprinting overview, 176, 184 parameters affecting outcomes, 176

628 potassium permanganate footprinting, see Potassium permanganate footprinting, ultraviolet C footprinting, see Ultraviolet C footprinting Intrinsic fluorescence, applications for DNA-binding proteins, 491, 493 DNA binding curve determination, data analysis, 497, 498 dissociation constant considerations for titration, 492, 490 materials, 494–495, 499 preliminary experiments, 495, 496, 499, 500 titration, 496, 497, 500 inner filter effects and correction of fluorescence, 493 origins, 492 principles of fluorescence, 491, 492 Isothermal titration calorimetry (ITC), see also Calorimetry, apparent heat change, 513, 514 dissociation constant determinations, 514, 515 effective heat change, 514 enthalpy of binding, 513–515, 518 principle, 513–515 Sox-5 HMG domain interaction with DNA, concentration determinations, complex, 528, 529 DNA, 527, 528 protein, 528 data analysis, 522, 524, 529, 530 enthalpy correction using differential scanning calorimetry, 525, 527 instrumentation, 520 materials, 520, 521, 529 titration, data acquisition, 521, 522, 529 range for DNA, 518

Index titration calculations, 515 ITC, see Isothermal titration calorimetry L Ligation-mediated polymerase chain reaction (LMPCR), violet C footprinting, see Ultraviolet C footprinting Linker histone-Fe(II) EDTA conjugate, cysteine substituted protein construction, ligation and transformation of polymerase chain reaction insert, 276, 280, 288 materials, 276 overexpression and purification, 276, 280, 281, 288 point mutation by polymerase chain reaction, 276, 279 rationale, 275, 276 reduction and modification with EDTA-2-aminoethyl 2pyridyl disulfide, 277, 278, 281, 282 linker histone function, 275 site-directed hydroxyl radical cleavage analysis, application, 286, 288 binding to reconstituted nucleosomes, 284, 285, 289 cleavage reaction, 285, 289 materials, 278, 279 Maxim–Gilbert G-specific reaction, 278, 284 nucleosome reconstitution, 278, 283, 284, 288 radiolabeling of DNA, 278, 282, 283, 289 sequencing gel, 279, 286 LMPCR, see Ligation-mediated polymerase chain reaction Lysine modification,

Index modifying reagents, 301 rationale for DNA-binding proteins, 301 reductive methylation with sodium cyanoborohydride, data analysis, 310 isotope incorporation, 302, 303 materials, 303, 304 overview, 301–303 peptide mapping, 308, 309, 313, 314 pulse–chase labeling, 306–309, 312, 313 quantification of modified residues, 306, 312 sodium cyanoborohydride recrystallization, 304 surface labeling of proteins and complexes, 305, 306, 311, 312 tritiated formaldehyde, determination of effective specific activity, 304, 305, 310, 311 M Methionine repressor, ethylation interference assay with MetJ, 230, 237–239 filter-binding assay using operator variants, 8, 9 Methylation protection/interference, dimethyl sulfate, DNA base reactivity, 221 interference assay, 225 materials, 222, 223 principles, interference assay, 222 protection assay, 221, 222 protection assay, 223–226 N Nitration, see Tyrosine nitration Nucleoprotein complex, limited proteolysis,

629 applications, 315, 316 materials, 318 overview, preliminary characterization of DNA-binding domains, 316 proteolysis, 316 purification of DNA-binding domain, 317 sequencing of protein, 317, 318, 320 protease selection, 316, 317 proteolysis conditions, 319, 320 purification of DNA-binding domain, 319–321 rationale, 315, 316 O Osmium tetroxide footprinting, advantages and limitations, 121, 122 applications, 122, 125, 127 safety, 128 materials, 122, 123, 128–130 stock solution preparation, 128–130 mechanism of thymidine attack, 130 reaction conditions, 123 detection of adducts, 123, 124, 130, 131 gel electrophoresis, 124 interpretation, 125, 127, 131, 132 ion effects, 125 in vivo modifications, 127, 128 P pBend vectors, see DNA bending PCR, see Polymerase chain reaction Peptide mapping, lysine modifications, 308, 309, 313, 314 RNA polymerase III after photoaffinity labeling, 365, 377, 378, 380 tyrosine nitrations, 295, 296, 298 Phage display, nucleic acid-binding proteins,

630 applications, 417 cloning into phage vector, 418, 421, 422, 426 enzyme-linked immunosorbent assay for binding, 419, 423, 424, 428 gene cassette library construction, 418, 420, 421, 425, 426 materials, 418, 419 overview, 417, 418 phage selection against nucleic acid targets, 418, 419, 422, 423, 427, 428 phage vector preparation, 418, 419, 424, 425 principle, 417 1,10-Phenanthroline-copper footprinting, advantages over other footprinting agents, 82, 83 chemistry of DNA cleavage, 79, 80, 82 complex isolation from free DNA, direct elution from gels, autoradiography, 95, 96 desalting, 97 excision, 96 extraction, 96, 97, 107 materials, 88–90 electrotransfer and elution from membrane, electrotransfer, 98 elution, 99 materials, 90, 91 principle, 97, 98 DNA structure and reaction rates, 80, 82 electrophoretic mobility shift assay coupling, benefits, 83, 85, 86 cleavage in gel, 94, 95, 106, 107 competition binding assay, 92, 104 dissociation rate determination, 92, 104, 105 gel preparation, 93

Index loading of gel, 94, 106 materials, 86, 87 optimization, binding reaction parameters, 103, 104 electrophoresis conditions, 104 exposure time to chemical nuclease, 92, 105, 106 preliminary assay, 92, 102 probe length, 103 preparative reaction, 93 principle, 82 running conditions, 94 in-gel cleavage, applications, 86 materials, 88 kinetic scheme for nuclease activity, 80, 81 RNA-binding protein analysis, 86 sequencing, autoradiography, 101, 102, 107, 108 gel loading and electrophoresis, 100, 101 ladder preparation, 91, 99, 100 reagents and equipment, 92 solutions, 91 Photoaffinity labeling, see also 8-Azidoadenine, overview of photolabeling groups, 323, 324 RNA polymerase II transcription complex, site-specific labeling, advantages and applications, 383, 384 materials, 384, 385 members of complex, 383 overview, 384 photocrosslinking, 388, 389, 391, 392 photoprobe preparation, AB-dUMP incorporation, 385, 388

Index annealing, 385 gel purification, 385, 387, 388, 391 primer extension, 385, 391 restriction digestion, 385 RNA polymerase III transcription complex, site-specific labeling, DNA probe synthesis, 364, 365, 371, 373, 380 DNA template immobilization, biotinylation, 369, 379 materials, 364 streptavidin bead binding, 369, 371, 379 nucleotide synthesis, AB-dUTP, 365–367, 378 dCTP analogs, 368, 369 materials, 364 varied photochemistry nucleotides, 368 varied tether-length nucleotides, 367, 368 peptide mapping, 365, 377, 378, 380 photoaffinity labeling, 365, 373, 376, 377, 380 Polymerase chain reaction (PCR), see also Ligation-mediated polymerase chain reaction, error-prone polymerase chain reaction for mutation introduction, 433, 436, 440, 447 phage display gene cassette library construction, 418, 420, 421, 425, 426 point mutation for cysteine substitution in histones, 276, 279 potassium permanganate footprinting application, 66, 70–72 systematic evolution of ligands by exponential enrichment, 603–604, 608–609

631 ultraviolet-laser footprinting analysis, 166–168 Potassium permanganate footprinting, advantages, 63, 64 applications, 64, 65 DNA modification, detection of modified bases, piperidine cleavage, 65, 66, 68, 69, 71 polymerase chain reaction amplification, 66, 70–72 primer extension, 66, 70–72 reaction mechanism, 64 in vitro experiments on linear DNA fragments, binding reaction, 68, 71 gel electrophoresis, 69, 71 modification reaction and stopping, 68, 71 piperidine cleavage, 68, 69, 71 radiolabeling of probe, 68 in vivo experiments, 69 materials, 67 Primer extension, see Polymerase chain reaction Proteolysis, see Nucleoprotein complex, limited proteolysis R Reconstitution, protein–DNA complexes for crystallization, annealing of DNA duplex, 551, 554 crystallization trials, 452, 553, 555 electrophoretic mobility shift assay, 551, 554 scale-up, 551, 552, 555 synthetic oligomer preparation, 550, 551, 554 TFIIIA recombinant protein purification, chromatography, 549, 552, 554 concentration determination, 549, 552, 554

Index annealing, 385 gel purification, 385, 387, 388, 391 primer extension, 385, 391 restriction digestion, 385 RNA polymerase III transcription complex, site-specific labeling, DNA probe synthesis, 364, 365, 371, 373, 380 DNA template immobilization, biotinylation, 369, 379 materials, 364 streptavidin bead binding, 369, 371, 379 nucleotide synthesis, AB-dUTP, 365–367, 378 dCTP analogs, 368, 369 materials, 364 varied photochemistry nucleotides, 368 varied tether-length nucleotides, 367, 368 peptide mapping, 365, 377, 378, 380 photoaffinity labeling, 365, 373, 376, 377, 380 Polymerase chain reaction (PCR), see also Ligation-mediated polymerase chain reaction, error-prone polymerase chain reaction for mutation introduction, 433, 436, 440, 447 phage display gene cassette library construction, 418, 420, 421, 425, 426 point mutation for cysteine substitution in histones, 276, 279 potassium permanganate footprinting application, 66, 70–72 systematic evolution of ligands by exponential enrichment, 603–604, 608–609

631 ultraviolet-laser footprinting analysis, 166–168 Potassium permanganate footprinting, advantages, 63, 64 applications, 64, 65 DNA modification, detection of modified bases, piperidine cleavage, 65, 66, 68, 69, 71 polymerase chain reaction amplification, 66, 70–72 primer extension, 66, 70–72 reaction mechanism, 64 in vitro experiments on linear DNA fragments, binding reaction, 68, 71 gel electrophoresis, 69, 71 modification reaction and stopping, 68, 71 piperidine cleavage, 68, 69, 71 radiolabeling of probe, 68 in vivo experiments, 69 materials, 67 Primer extension, see Polymerase chain reaction Proteolysis, see Nucleoprotein complex, limited proteolysis R Reconstitution, protein–DNA complexes for crystallization, annealing of DNA duplex, 551, 554 crystallization trials, 452, 553, 555 electrophoretic mobility shift assay, 551, 554 scale-up, 551, 552, 555 synthetic oligomer preparation, 550, 551, 554 TFIIIA recombinant protein purification, chromatography, 549, 552, 554 concentration determination, 549, 552, 554

632 materials, 548, 549 overview, 547, 548 vectors, 549 Restriction endonuclease, oligonucleotide assays, association rates of reaction components, 465, 484 dissociation constant, data analysis for determination, 468, 472, 485, 486 direct versus competition titration, 616, 557, 614 electrophoretic mobility shift assay for determination, competitive equilibrum binding, 481 data analysis, 482, 483 direct titration, 481, 482, 487, 488 materials, 471 filter binding assay, competitive equilibrium binding, 478–480, 487, 488 data analysis for competitive titration, 481 data analysis for direct binding, 478, 485, 486 direct titrations, 476, 477, 485–487 materials, 471 fluorescence anisotropy determination using hexachlorofluoresceinlabeled oligonucleotides, data analysis, 483, 484, 488 fluorescence measurements, 481, 488 materials, 470–472 overview, 469, 470, 486, 487 measurement techniques, 615, 616 range of values, 467, 468 equilibrum constant determination with fluorescence anisotropy,

Index 469, 486, 487 single turnover rate constant, components, 465, 466 measurement, data analysis, 471, 474, 476, 488 materials, 470, 471, 487 principle, 466, 484, 485 rapid-hydrolyzing enzymes, 473 slow-hydrolyzing enzymes, 472, 473, 484, 485, 488 specificity determination using structural perturbation approach, 465, 466 RNA polymerase–promoter interaction, bacterial holoenzyme structure, 339 hydroxyl radical interference, 248, 249 initiation complex formation, 339, 340 RNA polymerase II transcription complex, site-specific photoaffinity labeling, advantages and applications, 383, 384 materials, 384, 385 members of complex, 383 overview, 384 photocrosslinking, 388, 389, 391, 392 photoprobe preparation, AB-dUMP incorporation, 385, 388 annealing, 385 gel purification, 385, 387, 388, 391 primer extension, 385, 391 restriction digestion, 385 RNA polymerase III transcription complex, site-specific photoaffinity labeling, DNA probe synthesis, 364, 365, 371, 373, 380 DNA template immobilization,

Index biotinylation, 369, 379 materials, 364 streptavidin bead binding, 369, 371, 379 nucleotide synthesis, AB-dUTP, 365–367, 378 dCTP analogs, 368, 369 materials, 364 varied photochemistry nucleotides, 368 varied tether-length nucleotides, 367, 368 peptide mapping, 365, 377, 378, 380 photoaffinity labeling, 365, 373, 376, 377, 380 site-specific protein–DNA photocrosslinking, DNA preparation, annealing, extension, and ligation, 348, 349, 357 chemical derivatization, 347, 348, 357 digestion and gel purification, 349, 350, 357 materials, 340, 342, 343, 356, 357 phosphorothioate oligodeoxyribonucleotide preparation, 346, 347 purification with reversedphase high-performance liquid chromatography, 348, 357 radiolabeling, 348, 357 intermediate complex preparation, 354, 355, 358 nuclease digestion of complex and gel analysis, 356 open complex preparation, 355, 358 photocrosslinking in-gel, N,N’-bisacryloylcystamine synthesis, 353, 354

633 excision and extraction of crosslinked complex, 356, 358 gel preparation, 354, 358 irradiation, 355, 356, 358 materials, 345, 346 RNA polymerase from bacteria, crude subunit and fragment preparation, 351, 352 histidine-tagged a-subunit preparation, 350, 351, 357 materials for preparation, 343–345 nickel affinity chromatography, 353 reconstitution, 352, 353 split derivatives, 340 transcription factor assays, see Transcription factor S Scanning transmission electron microscopy (STEM), advantages for DNA–protein complex studies, 589, 598, 599 complex classification, 590 crosslinking of DNA–protein complexes, 591, 599 image analysis, 595–598, 600 materials for DNA–protein complex imaging, additives, 591, 600 buffers, 591, 592, 600 films, 592, 600, grids, 592 water, 591, 599 microscope operation, 594, 595, 600 molecular weight determination, 589 resolution, 590, 599 specimen preparation, concentrations of complexes and components, 593

634 fixation, 591, 594 polylysine-pretreated grids, 594, 600 wet film, hanging drop method, 593, 594, 599, 600 SELEX, see Systematic evolution of ligands by exponential enrichment Singlet oxygen footprinting, detection of reaction sites, 157, 159 eosin–Tris complex preparation, 156 instrumentation, 154 irradiation conditions, 156, 157 materials, 154–156 nucleoprotein complex formation, 158 overview, 152 rationale and advantages, 151, 152 reaction with DNA, diffusion, 152, 153 half-life of singlet oxygen, 154 rate of reaction and DNA structure, 153, 154 Site-specific protein–DNA photocrosslinking, applications, 339 overview, 337, 338 RNA polymerase–promoter interactions, DNA preparation, annealing, extension, and ligation, 348, 349, 357 chemical derivatization, 347, 348, 357 digestion and gel purification, 349, 350, 357 materials, 340, 342, 343, 356, 357 phosphorothioate oligodeoxyribonucleotide preparation, 346, 347 purification with reversedphase high-performance liquid chromatography, 348, 357 radiolabeling, 348, 357

Index intermediate complex preparation, 354, 355, 358 nuclease digestion of complex and gel analysis, 356 open complex preparation, 355, 358 photoaffinity labeling, see Photoaffinity labeling photocrosslinking in-gel, N,N’-bisacryloylcystamine synthesis, 353, 354 excision and extraction of crosslinked complex, 356, 358 gel preparation, 354, 358 irradiation, 355, 356, 358 materials, 345, 346 RNA polymerase from bacteria, crude subunit and fragment preparation, 351, 352 histidine-tagged a-subunit preparation, 350, 351, 357 materials for preparation, 343–345 nickel affinity chromatography, 353 reconstitution, 352, 353 split derivatives, 340 validation with crystal structures, 339 Sodium cyanoborohydride, see Lysine modification Southwestern blot, applications, 256 DNase I footprinting combination, alternative cleavage agents, 137 blotting, alignment markers, 144, 148 autoradiography, 144, 148 electroblotting, 143, 147 gel electrophoresis, 143, 147 overview, 142, 143 probing with DNA, 143, 147, 148 reagents and equipment, 140, 141, 147 solutions, 137–140, 146, 147

Index DNase I treatment of blots, extraction, 145 gel electrophoresis and autoradiography, 146, 148 reaction conditions, 144, 145, 148 reagents and equipment, 142 solutions, 141, 142 fidelity, 137 rationale and advantages, 135–137 identification of DNA-binding proteins, electroblotting, 259–261 extract preparation, 256, 260 gel electrophoresis, 258, 259, 261 materials, 256, 258, 260, 261 membrane probing, 260, 262 overview, 135, 255, 256 principle, 255 Sox-5 HMG domain, differential scanning calorimetry of interaction with DNA, concentration determinations, complex, 528, 529 DNA, 527, 528 protein, 528 correction of isothermal titration calorimetry-derived enthalpies, 525, 527 data acquisition, 524, 530, 531 data analysis, 525 instrumentation, 520 materials, 520, 521, 529 dissociation constant determination for DNA, 518 DNA sequence specificity, 516 isothermal titration calorimetry of interaction with DNA, concentration determinations, complex, 528, 529 DNA, 527, 528 protein, 528 data analysis, 522, 524, 529, 530

635 enthalpy correction using differential scanning calorimetry, 525, 527 instrumentation, 520 materials, 520, 521, 529 titration, data acquisition, 521, 522, 529 range for DNA, 518 structure, 512 ultraviolet melting curve for DNA complex, 518, 519 SPR, see Surface plasmon resonance STEM, see Scanning transmission electron microscopy Surface plasmon resonance (SPR), Biacore instrument principles, 535–537, 541 binding curve analysis, kinetic analysis, 539–541, 544, 545 stoichiometry and equilibrium analysis, 538, 539 consistency tests, 557 DNA immobilization, immobilization reaction, 543, 544 overview, 537 streptavidin coupling, 542, 544 materials for DNA–protein binding analysis, 541 protein binding to immobilized DNA, 537, 538, 543, 545 recapture, 545, 546 refractive index relationship to mass, 535, 541, 544 Systematic evolution of ligands by exponential enrichment (SELEX), applications for nucleic acid-binding proteins, 603, 604 complex formation and washing, 607–609 DNA template and primers, 605, 608, 609 in vitro transcription, 606 materials, 605

636 nickel bead binding of histidinetagged proteins, 606, 609 overview, 603–605 partitioning matrix preparation, 606, 608 polymerase chain reaction, 605, 606–609 reverse transcription of RNA samples, 607 rounds of selection, 607, 609 T Tetranitromethane (TNM), see Tyrosine nitration TNM, see Tetranitromethane Transcription factor, functions, 447 initiation complex formation, 339, 340 RNA polymerase II transcription complex, 383 TFIIIA recombinant protein purification, chromatography, 549, 552, 554 concentration determination, 549, 550, 554 materials, 548, 549 overview, 547, 548 vectors, 549 transcriptional activation assays, abortive initiation, data analysis, 457, 461, 462 dinucleotide primer, 455, 461 fluorescence detection, 461 incubation conditions, 456 paper chromatography, 455–457 principal, 448 materials, 452, 454, 557, 458 overview, 451–452 transcript assays, binding reaction, 454, 455, 457–459

Index electrophoretic analysis, 455, 460, 461 principle, 452 transcription reaction, 455, 459, 460 Transmission electron microscopy, see Electron microscopy Tryptophan fluorescence, see Intrinsic fluorescence Two-dimensional crystallization, advantages over X-ray crystallography, 557 crystallization conditions, 562, 563, 564, 567 electron microscopy, crystal transfer to grid, 563, 564–567 evaluation of specimens, 564, 567 negative staining, 563, 564, 566 support preparation, 560–562 image analysis, 565, 566 lipid–protein interactions, 557–560, 564, 566 materials, 560 Two-wavelength femtosecond laser irradiation, see Ultraviolet laserinduced protein–DNA crosslinking Tyrosine fluorescence, see Intrinsic fluorescence Tyrosine nitration, accessibility studies, 292, 293 functional studies, 293, 296 kinetic analysis, 293 materials, 294 modifying reagents and tyrosine specificity, 291, 292 peptide mapping, 295, 296, 298 rationale for DNA-binding proteins, 291 tetranitromethane nitration reaction, 295, 296, 298 U Ultraviolet C footprinting, ligationmediated polymerase chain

Index reaction for in vivo footprinting, advantages and limitations, 185, 187 cleavage for DNA sequencing productss, A reaction, 201 C reaction, 201, 202 G reaction, 201 overview, 200, 201 processing of samples, 202 reagants, 192 T+C reaction, 201 DNA polymerase selection, 191 DNA purification, DNA extraction, 200, 213 materials, 191, 192 nuclei isolation, 200 quantification, 200, 213 gel electrophoresis and electroblotting, blotting, 209, 214 electrophoresis, 208, 209 materials, 196, 197 hybridization, digoxigenin-labeled probe, 197, 198, 210, 214, 215 materials, 197 radiolabeled probe, 197, 209, 214 information from photofootprints, 185, 186 instrumentation, 193 ligation, ligation reaction, 207 materials, 195 modified bases and conversion to single-strand breaks, 186, 188, 194, 205, 206 overview, cyclobutane pyrimidine dimer formation, 178 pyrimidine (6–4) pyrimidone photoproduct, 180 photoproduct distribution, 183, 185 polymerase chain reaction, cycles, 207, 208

637 materials, 195, 196, 213 primer extension, incubation conditions, 206, 207 materials, 194, 195, 212, 213 single-stranded hybridization probe preparation, amplification product purification and quantification, 198, 199, 211, 215 digoxigenin labeling, 199, 212, 215 length, 210, 215 materials, 198, 199, 213 polymerase chain reaction amplification, 198, 210, 211 radiolabeling, 199, 211, 212 ultraviolet irradiation, 203, 213, 214 Ultraviolet crosslinking of DNA– protein complexes, see 8Azidoadenine; Site-specific protein–DNA photocrosslinking; Ultraviolet laser-induced protein– DNA crosslinking Ultraviolet-laser footprinting, advantages over other footprinting techniques, 161, 163 binding reaction, 165, 167, 168 disadvantages, 164 in vivo footprinting, 167, 169, 172 instrumentation, 164 integration host factor/yjbE interaction analysis, 164–167 kinetic analysis, 163 laser operation, 165–167 materials, 164, 165 photoreactions, 163 primer extension, 166–168 principle, 161–163 sequencing and interpretation, 166, 168, 169 troubleshooting, 168, 169 Ultraviolet laser-induced protein–DNA crosslinking, advantages, 395

638 applications, 396 histone–DNA complexes, DNA hybridization for sequence identification, 400, 401 dot immunoassay, 399 immunoprecipitation, 399–401 isolation of crosslinked complexes, 398, 399, 400 instrumentation, 397, 400 irradiation techniques, 398, 400 materials, 397, 398 mechanism, 395, 396 overview, 396, 397 two-wavelength femtosecond laser irradiation, applications, 612, 615, 616 DNA integrity checking, 615, 616 electrophoretic mobility shift assay for optimization, 614, 615 in vitro crosslinking, 614 in vivo crosslinking, 614, 616 lasers, 616, 616 principal, 612 rationale, 611, 612 reagents and solutions, 613 yield determination for crosslinks, 615, 616 Uranyl photofootprinting, applications, 111–113, 115 binding reaction, 112, 116, 117 cleavage reaction, 113, 117 comparison with other footprinting techniques, 113, 115 gel electrophoresis and autoradiography, 113, 117

Index hypersensitive cleavage sites, 115 interference probing by phosphate ethylation, 115 l-repressor/OR1 complex analysis, 113 materials, 112, 116, 117 mechanism of photocleavage, 112 phosphate probing on DNA backbone, 113, 115 principle, 111 Y Yeast reporter assay of DNA–protein interactions, activation domains, 433, 446 biochemical analysis of mutants, 444 error-prone polymerase chain reaction for mutation introduction, 433, 436, 440, 447 expression plasmid, 432, 433, 435, 438, 439, 446, 447 β-galactosidase assay, 437, 439, 440, 443, 444, 448 initial design and testing, 435, 437–440 materials, 435–437 optimization, 446 overview, 431–434 reporter plasmid, 432, 445, 435, 437, 438, 446 screening for mutants, 437, 441–443, 448 sequence analysis, 444 transformation and homologous recombination, 436, 437, 440, 441, 447, 448 yeast strains, 435, 444, 445

CANCER DRUG DISCOVERY AND DEVELOPMENT Series Editor: Beverly A. Teicher

Tumor Models in Cancer Research Edited by

Beverly A.Teicher Lilly Research Laboratories, Indianapolis, IN Cancer researchers have made significant progress over the years by developing appropriate and accurate animal disease models, the most important being transplantable rodent tumors. In Tumor Models in Cancer Research, Beverly A. Teicher and a panel of leading experts comprehensively describe for the first time in many years the state-of-the-art in tumor model research. The wide array of model systems detailed form the basis for the selection of both compounds and treatments that go into clinical testing of patients, and include syngeneic, human tumor xenograft, orthotopic, metastatic, transgenic, and gene knockout models. These models represent the efforts of many investigators over the years and approach, with increasing precision, examples that serve as guides for the selection of agents and combinations for the treatment of human malignancy. Synthesizing many years of experience with all the major in vivo models currently available for the study of malignant disease, Tumor Models in Cancer Research provides preclinical and clinical cancer researchers alike with a comprehensive guide to the selection of these models, their effective use, and the optimal interpretation of their results. 䊏 Reviews the state-of-the-art of the in vivo tumor models available for cancer research 䊏 Discusses the use and interpretation of tumor models and endpoints 䊏 Covers metastatic models, including syngeneic, orthotopic, and GFP-labeled tumor models 䊏 Includes a comprehensive bibliography for each in vivo tumor model

Contents Part I: Introduction. Perspective on the History of Tumor Models. Part II: Transplantable Syngeneic Rodent Tumors. Murine L1210 and P388 Leukemias. Transplantable Syngeneic Rodent Tumors: Solid Tumors of Mice. B16 Murine Melanoma: Historical Perspective on the Development of a Solid Tumor Model. Part III: Human Tumor Xenografts. Xenotransplantation of Human Cell Cultures in Nude Mice. GFP-Expressing Metastatic-Cancer Mouse Models. Human Tumor Xenografts and Explants. Part IV: Carcinogen-Induced Tumors: Models of Carcinogenesis and Use for Therapy. Hamster Oral Cancer Model. Mammary Cancer in Rats. Carcinogen-Induced Colon-Cancer Models for Chemoprevention and Nutritional Studies. Part V: Mutant, Transgenic, and Knockout Mouse Models. Cancer Models: Manipulating the Transforming Growth Factor-` Pathway in Mice. Cyclin D1 Transgenic Mouse Models. Mice Expressing the Human Carcinoembryonic Antigen: An Experimental Model of Immunotherapy Directed at a Self, Tumor Antigen. The p53-Deficient Mouse as a Cancer Model. The Utility of Transgenic Mouse Models for Cancer Prevention Research. Part VI: Metastasis Models. Metastasis Models: Lungs, Spleen/Liver, Bone, and Brain. Models for Evaluation of Targeted Therapies of Metastatic Disease. Part VII: Normal Tissue Response Models. Animal Models of Oral Mucositis Induced by Antineoplastic Drugs and Radiation. The Intestine as a Model for Studying Stem-Cell Behavior. SENCAR Mouse-Skin Tumorigenesis Model. Murine Models of Bone-Marrow Transplant Conditioning. Anesthetic Considerations for the Study of Murine Tumor Models. Part VIII: Disease and Target-Specific Models. Tissue-Isolated Tumors in Mice: Ex Vivo Perfusion of Human Tumor Xenografts. Human Breast-Cancer Xenografts as Models of the Human Disease. Animal Models of Melanoma. Experimental Animal Models for Renal Cell Carcinoma. Animal Models of Mesothelioma. SCID Mouse Models of Human Leukemia and Lymphoma as Tools for New Agent Development. Models for Studying the Action of Topoisomerase-I Targeted Drugs. Spontaneous Pet Animal Cancers. Part IX: Experimental Methods and End Points. In Vivo Tumor Response End Points. Tumor-Cell Survival. Apoptosis In Vivo. Transparent Window Models and Intravital Microscopy: Imaging Gene Expression, Physiological Function, and Drug Delivery in Tumors. Index.

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Cancer Drug Discovery and Development™ TUMOR MODELS IN CANCER RESEARCH ISBN: 0-89603-887-4 humanapress.com

9 780896 038875

DNA'Protein Interactions: Principles and Protocols

DNA'Protein Interactions: Principles and Protocols

DNA'Protein Interactions: Principles and Protocols